Development of placenta

Embryo

Cleavage 2

Cleavage

Fertilizes egg

Follicle development

Fertilization

Anatomy of placenta

Prenatal Diagnosis of Chromosomal Anomalies /DR. Alaa Mosbah , MD OBS & GYN

Introduction : Prenatal diagnosis of chromosomal anomalies employs a variety of techniques either as a screening procedure for relatively prevalent disorders or as a diagnostic procedure for known familial conditions. The former identifies an increased likelihood of a fetal abnormality in an apparently normal pregnancy, whereas the latter confirms or refutes the existence of an actual anomaly in a fetus believed to be at increased risk . Currently available prenatal non-invasive screening tests are ultrasonography and various biochemical tests, while CVS, amniocentesis, and fetal blood sampling are invasive diagnostic procedures. High-risk pregnancies : include the following 1. Advanced maternal age (age 35 years or more at estimated date of delivery). Some authors also consider paternal age of approximately 50 years or older. 2. Family history (self, spouse, child, parent or sibling) of previous birth of a child with chromosome abnormality / multiple structural defects / ONTDs, or metabolic disorder. 3. Suspicious biochemical results. 4. Abnormal morphological scan. 5. Parent with a chromosome disorder. 6. Parent who are carriers of specific genetic defects such as cystic fibrosis, Tay-Sach dystrophy, sickle cell anemia, Falconis anemia&thalassaemia The most common reason for prenatal diagnosis of chromosome abnormalities is to look for evidence of trisomies, Turner syndrome and triploidy. Trisomy 13, 18, and 21 are the most common, with trisomy 21 comprising about half of all the trisomies identified. Various screening and diagnostic procedures used to detect common chromosomal abnormalities are discussed here. Screening procedures : Non invasive diagnostic procedures : 1.Ultrasonography Ultrasound scanning is offered to all pregnant women. It is non-invasive and has no inherent procedure related loss. First-trimester sonography is offered mainly to confirm the gestational age, to identify singleton or multiple pregnancy and to measure nuchal thickness (NT). Second-trimester ultrasound is generally recommended at 19 to 21 weeks to detect fetal structural defects. Nuchal translucency (NT) : NT refers to the subcutaneous space between the skin and the cervical spine in the fetus. The ultrasonographic findings of NT in the first-trimester of the pregnancy reportedly has been associated with fetal chromosome abnormalities including trisomies 21,18,13 and triploidy and Turner syndrome (45XO). Among karyotypically normal fetuses increased NT may be related to certain birth defects including cardiac septal defects, diaphragmatic hernia, renal defects, omphalocele, body-stalk anomaly, fetal akinesia syndrome, and certain skeletal dysplasias. Certain genetic defects and infections may also lead to increased NT. Various genetic defects associated with increased NT are Arthrogryposis, Noonan's syndrome, Smith-Lemli-Opitz syndrome, Stickler syndrome, Jarco-Levine syndrome, Miller-Dieker syndrome. Increased NT may be found in a normal fetus. Therefore, increased NT does not mean that the fetus is chromosomally abnormal but it does mean an increased risk for some disorders and birth defects. NT measurements can be taken from the sagittal section of the fetus usually used to obtain the crown-rump length, or from a transverse suboccipitobregmatic view of the fetal head. NT values are crown-rump-length-dependent. Measurements equal to or more than 3mm in the first-trimester and more than 6mm in the second-trimester are considered as abnormal. NT is apparently a powerful tool for the detection of aneupolidy, particularly Down's syndrome. For isolated nuchal edema, the risk for trisomy 21 may be 15 times the background. The estimated detection rate for Down's syndrome using NT at 10 to 14 weeks of gestation combined with maternal age is about 80% at a cut off risk of 1 in 300 or higher. However, there is considerable disagreement in the literature as to the precise Down's syndrome detection rate that can be expected with this form of sonography. Cystic hygroma I s caused by the malformation of the fetal lymphatic system and is found commonly in fetuses with Monosomy X (Turner syndrome), and other chromosomal anomalies. An important part of evaluation of nuchal membrane is the evaluation of the subtle signs of hydrops fetalis. Even in the presence of a normal karyotype, perinatal outcome is very poor in the presence of fetal hydrops. Choroid plexus cyst (s) Vast majority of cyst (s) is benign and usually disappears by 24-25 weeks. However, these cysts may be associated with chromosomal anomalies, primarily trisomy 18 but occasionally trisomy 21. Choroid plexus cysts have been reported to be associated with trisomy 18 in 1% to 6 % cases. For isolated choroid plexus cysts, the risk for trisomy 18 and trisomy 21 is about 1.5 times the background. Intracardiac echogenic foci Increased echogenicity (golf ball) located in chordae tendinae is seen in 3 to 5 % of fetuses and is considered by most investigators of no pathological significance. However, they are sometimes associated with chromosomal abnormalities particularly, trisomy 21. For isolated hyperechogenic foci the risk for trisomy 21 may be four times the background. Hyperechogenic fetal bowel Hyperechogenic bowel has been described as a normal variant, but may also be associated with cystic fibrosis, meconium peritonitis, cytomegalovirus infection or trisomy 21. For isolated hyperechogenic bowel, the risk for trisomy 21 may be three times the background. Mild hydronephrosis Minimal pyelectasis in the fetus is common and unlikely to be significant in every case. However, UPJ obstruction and reflux may manifest initially as mild fetal pyelectasis. In addition, karyotype abnormalities, remarkably trisomy 21, are reported to be associated with mild pyelectasis. Long bone biometry Short humeri and short femurs may be found in fetuses with Down's syndrome. Measurements are usually compared with BPD rather than menstrual age due to the uncertainty of the menstrual history. If the femur length is below 5th centile and all other measurements are normal, the fetus is likely to be normal but rather short. However, this finding is rarely related to dwarfism and occasionally trisomy 21. Fetal hand Abnormalities of fetal hand such as polydactyly, over-riding fingers, or abnormal hand positioning, especially if associated with polyhydramnios, have been reported to be associated with fetal chromosomal abnormalities. Club foot / Rocker bottom feet (positional deformities) Club foot has been associated with a variety of chromosomal abnormalities, especially trisomy 13 and 18. Whether or not infants with isolated club foot are at significantly increased risk of chromosomal abnormalities is still unresolved. Rocker bottom feet are also a feature of trisomy 13 and 18. The finding of this deformity should be a stimulus to detailed fetal examination and fetal karyotyping. Single umbilical artery A single umbilical artery is found in 0.5% to 2% of newborns and is associated with structural or other anomalies in 20% to 50% of cases; usually relatively minor anomalies of the renal or genital tracts. Whether or not the presence of an isolated single umbilical artery is an indication for genetic studies is still uncertain. Intrauterine growth restriction A significant percentage of fetuses with chromosomal abnormalities are growth restricted. Some investigators have suggested the presence of IUGR alone is an indication for genetic studies. Many additional abnormalities such as cerebellar hypoplasia, isolated pericardial effusion, sandal-foot, reduction defect of forearm, low set ears or shortened ear length, fetal cholecystomegaly, and polyhydramnios have been found to be associated with chromosomal anomalies. When these findings are seen in isolation, it is still controversial whether or not amniocentesis should be offered. The sensitivity of ultrasonography for detection of fetal trisomic conditions varies with the type of chromosome abnormality, gestational age at the time of sonography, reasons for referral, criteria for positive sonographic findings, and the quality of the sonography. As an estimate, 1 or more sonographic findings can be identified in approximately 90% of fetuses with trisomy 13, 80% of fetuses with trisomy 18, and 50 % to 70% of fetuses with trisomy 21. Clusters of minor sonographic markers greatly increase the likelihood of karyotypic abnormality compared with a single minor marker . Cardiovascular anomalies Since malformations of the heart are the most common birth defect in fetuses with serious chromosomal abnormalities, color Doppler ultrasound evaluation of the cardiovascular system can be used to improve the detection rate of these abnormalities. By the inclusion of color Doppler sonography in genetic ultrasound, 96% of fetuses with Down's syndrome, 98% of all trisomies, and 88 % of all chromosomal abnormalities can be detected . A complete normal ultrasound examination result reduces the risk of abnormal karyotype,by,62%. 2.Biochemical,tests A large number of serum analytes have been found to be associated with chromosomal abnormalities. Various principal biochemical analytes currently available for the prenatal screening of chromosomal abnormalities during first and second-trimester of pregnancy are; pregnancy associated plasma protein-A (PAPP-A), maternal serum alpha-feto-protein (MSAFP), free-beta hCG, total hCG, unconjugated estriol (uE3), and inhibin-A. PAPP-A and free-beta hCG are only biochemical markers identified to be of value in first-trimester screening. Free-beta hCG is the only marker that is effective in both the first and second-trimester pregnancy. PAPP-A It is a homotetrameric glycoprotein (metzincins family of metalloproteases) which is synthesized in chorionic villi [35]. It can be measured in maternal serum; its concentration increases rapidly after 7th week of pregnancy and its potential clinical usefulness is greatest in the first trimester (10th to 14th week of gestation). When the fetus is affected with trisomy 21, PAPP-A levels are decreased by more than half [36]. Low PAPP-A is also associated with trisomy 18 and 13. Using PAPP-A alone, detection rate of Down's syndrome is about 40%. When PAPP-A was combined with maternal age, the detection rate increased to 50% with a 5% screen-positive rate [37]. Decreased production of PAPP-A may cause intrauterine growth restriction [38] as this hormone control the level of insulin -like growth factors (IGF) in the placenta. MSAFP MSAFP is a glycoprotein produced by the fetal yolk sac and fetal liver. Fetal plasma concentration increases to a maximum (approximately 3.0-4.0 g/L) between 13-14 weeks of gestation. Maternal serum levels peak at about 30 weeks (about 250 mg/L). After birth, maternal and infant AFP rapidly declines. High MSAFP levels may occur in the following conditions; 1. Underestimated gestational age 2. Multiple gestation. 3. Neural tube defects (anencephaly, spina bifida, encephalocele). 3 Abdominal wall defects (omphalocele, gastroschisis) 4. Various other fetal abnormalities such as hydrocephaly, microcephaly, cystic hygroma, cyclopia, tetralogy of Fallot, duodenal atresia, congenital nephropathies, sacrococcygeal teratoma, hydrops fetalis and Turner's syndrome without hygroma. 5. Complications of pregnancy ( fetal distress, growth retardation, early intrauterine death, placental defects, abdominal pregnancy, and maternal proteinuric preeclampsia). 6. Congenital fetal neoplasms, and maternal AFP producing neoplasms. Low MSAFP levels may be associated with 1. Incorrect pregnancy dating (less advanced than originally thought). 2. Complications of pregnancy (missed abortion, spontaneous abortion, late fetal death, molar pregnancy, choriocarcinoma). 3. Some normal pregnancies. 4. Large-for-dates. Very low MSAFP predicts an unusually high rate of large birth weight infants, with increased fetal, intrapartum, and neonatal consequenses. 5. MSAFP less than the median may indicate an increased risk of chromosomal abnormalities such as trisomy 21 and trisomy 18. The level of MSAFP in Down's syndrome pregnancies are about 72% of the normal values for weeks 14 to 21. Using maternal age and MSAFP level, the detection rate of Down's syndrome pregnancies is about 25-33%, at a false positive rate of 5% . Total hCG hCG is a dimeric glycoprotein composed of two non-covalently linked polypeptide sub-units, alpha and beta. It is first secreted by the fertilized ovum and later by placental tissue. Serum hCG levels increase exponentially between 3-10 weeks of pregnancy. Levels reach a peak during the first-trimester (about 100000 m IU/ml) and decline during the second and third-trimesters. In normal second-trimester maternal sera, the level of intact hCG ranges from 20,000-50,000 mIU/ml. In trisomy 21 pregnancies, second -trimester hCG levels are elevated varying from 2.04 to 2.5 MoM or greater while in trisomy 18 and 13, hCG levels are lower than normal. Using maternal age and hCG levels, Down's syndrome detection rate is about 60 % at a false positive rate of 6.7 % . Very high levels of hCG suggest trophoblastic disease. Various workers found a significant increase in the levels of free beta-hCG in trisomy 21, and concluded that this biochemical analyte is superior to intact hCG for the detection of trisomy 21. The levels of this analyte are reduced in the blood of women carrying fetuses with trisomy 18 . Unconjugated estriol (uE3) The substrate for estriol begins as dehydroepiandrosterone (DHEA) made by the fetal adrenal glands. This is later hydroxylated in fetal liver and cleaved by steroid sulphatase in placenta where unconjugated fraction converts to uE3. The amount of estriol in maternal serum is dependent upon a viable fetus, a properly functioning placenta, and maternal wellbeing. In normal pregnancies, uE3 levels increase from about 4 nmol/L at 15 weeks gestation to about 40 nmol/L at delivery. uE3 tends to be lower when trisomy 21or 18 is present and when there is adrenal hypoplasia with anencephaly. Second-trimester maternal serum uE3 levels in Down's syndrome pregnancies are approximated 75% of the values expected in normal pregnancies . Using maternal age and uE3 levels detection rate of about 45.7% at a false positive rate of 9.1 % was found . Inhibin-A Inhibin-A is a heterodimeric glycoprotein of placental origin similar to hCG. Inhibin-A levels in maternal serum are relatively constant through the 15th-18th week of pregnancy. Maternal serum levels of inhibin-A are twice as high in pregnancies affected by Down's syndrome as in unaffected pregnancies while in trisomy 18, inhibin-A levels are lower than normal. Inhibin-A is used with three other analytes (MSAFP, hCG, uE3) and maternal age to characterize more accurately Down's syndrome risk, and a reduction of the false positive rate was detected. Currently available evidences suggest that sensitivity of individual maternal serum markers is substantially low. To improve the sensitivity and specificity of biochemical markers, various combinations of 2, 3, or 4 analytes are offered during first and / or second-trimester screening. Various combinations of analytes include; 1. First-trimester maternal serum double test using PAPP-A and free-beta hCG. This is vastly more effective than any other screening program and is of great advantage because it is available in the first-trimester. The disadvantage is that the test will not detect neural tube defects, so a MSAFP test will still need to be done. After combining PAPP-A and free beta-hCG with maternal age, detection rate of aneuploidy is about 60% at a 5% screen-positive rate. 2. Second-trimester screening includes; double test (MSAFP+ free-beta hCG or total hCG), triple test (MSAFP+hCG+uE3), and quad screen test (MSAFP+hCG+uE3+inhibin-A). When an ultrasound scan is used to estimate gestational age the Down's syndrome detection rate for a 5% false positive rate is estimated to be 59% using the double test (AFP and hCG), 69% using the triple test (AFP,hCG,uE3), and 76% using the quad test (AFP,hCG,uE3, inhibin-A), all in combination with maternal age. Main disadvantage of second-trimester screening is the timing of the test. By the time a definitive diagnosis is made via amniocentesis (after a positive screen result), the option of termination of pregnancy can be difficult. MSAFP is always included as a component of second-trimester biochemical screening because this analyte is widely used for the detection of ONTDs at this stage of pregnancy. Addition of uE3 as an analyte for prenatal screening is controversial. Some authors pointed out that inclusion of this analyte does not improve detection rates, where as in another study a fall in detection rate was noticed. On the other hand, some workers rely on the usefulness of uE3 as a serum marker. In a study, it was found that double test is not worse than the triple test and the double test is preferred because of lower running cost. Comparing free beta-hCG and total hCG, it was found that free beta hCG is better option because it improves the Down's syndrome detection efficiency by 10 % over total hCG at a lower false positive rate. Review of the literature suggests that the combined use of free beta-hCG and AFP instead of total hCG, AFP and uE3, is better screening test because it is more effective, less expensive, and is not limited to 16-20 weeks gestation. The addition of uE3 and inhibin-A adds no significant advantage to the double test. However, some workers recommend triple test while some rely on quad test for better results. Combined screening Since maternal biochemical and sonographic markers are largely independent, combined risk estimate results in even higher detection rate than either alone. Thus combination of a multiple marker test (PAPP-A and free b-hCG) and an ultrasound at 11 to 14 weeks which is targeted to look NT, and maternal age gives a detectable rate of aneuploidy of about 90 % with an invasive testing rate of about 5%. But in many high-risk situations this may still leave the patient at risk. Integrated screening The integrated test includes measurements of PAPP-A and NT in the first-trimester and measurements of MSAFP, uE3, hCG, and inhibin-A in the second-trimester. The integrated test can achieve a high detection rate with a much lower false positive rate (0.9%) than screening based on markers measured in either trimester alone. Consequently the need for amniocentesis or CVS is reduced by four fifths, with a similar reduction in the loss of unaffected fetuses. The estimates of the integrated test's performance are based on there being little or no correlation between the first and second-trimester markers used with the exception of free beta-hCG and total hCG. Disadvantage of the test is that it is expensive, and option of making the diagnosis in the first-trimester is eliminated. Recently, Azuma M, et al used Lens culinaris agglutinin reactive alpha-fetoprotein ratio for the detection of fetal Down's syndrome in combination with traditional serum markers such as AFP, hCG and uE3 and found a detection rate of 83.9% with a 5.1% false positive rate. Various factors affect the accuracy of biochemical tests. Therefore, adjustments are made to take account of these factors when categorizing the result as screen positive or screen negative. These factors are: 1. Gestational age: Measured concentrations of the serum markers vary with gestational age, Between 9 to 14 weeks, maternal serum free beta-hCG decreases, median free beta-hCG also decreases while median PAPP-A increases. Between 14-22 weeks, Median MSAFP and median uE3 increases, median hCG decreases while median inhibin-A is less affected. 2. Maternal weight: Maternal weight has a significant effect on the screening process. There is inverse correlation between body weight and concentration of biochemical markers. If the volume of distribution is greater, the concentration of AFP will be smaller. Maternal weight also has a significant inverse correlation on inhibin-A levels. PAPP-A and free beta-hCG also show a small but significant negative correlation. But there is no correlation for uE3. 3. Smoking: Maternal smoking has a small effect on overall screening performances. Women who smoke are more likely to produce a small amount of PAPP-A. 4. Effect of parity: Parity significantly affects the mean MoM of hCG but did not affect the values for uE3 or MSAFP. 5. Multiple pregnancies: On an average, the levels of MSAFP and free beta-hCG were twice as high in twins and over three times as high in triplets. 6. Difference between races of ethnic groups: Medians are significantly different in races of ethnic groups. Black women, on an average, have higher levels of MSAFP than Caucasian, Asian or Hispanic women. 7. Insulin-dependent diabetes mellitus: MSAFP levels in IDDM have shown an approximately 20% decrement while uE3 and inhibin-A levels are reduced to a smaller extent . Conclusions of multiple marker screening test: 1. Maternal blood screening is originally developed for pregnant women who are at a 'low-risk' for disorders being screened. However, in certain situations maternal serum marker screening can be assayed in women > or =35 years.. 2. Gestational age must be accurately known. First-trimester screening is offered between 10 to 14 weeks of gestation, while second-trimester screening tests are given between 14 and 22 weeks of pregnancy, with 16 to 18 weeks (when the hormone levels are most consistent) being the optimum time. Recently Muller F, et al assessed the diagnostic value of maternal serum marker screening at 18-35 weeks and concluded that biochemical screening is feasible at 18 weeks and later, which may be of interest in selected cases. 3. Multiple gestation screening is not as sensitive as singleton screening with biochemical analysis. 4. Screening utilizing biochemical test, NT and maternal age mainly identifies aneuploidy and some other chromosomal abnormalities only and therefore shall miss majority of other non-Down, non-trisomy 18 chromosome abnormalities, nor does it identify other physical or mental birth defects. 5. A low PAPP-A, low MSAFP, low uE3, and high free beta-hCG, and high inhibin-A levels are associated with higher risk for Down's syndrome while low levels of all hormones suggest an increased risk for trisomy 18. In trisomy 13, PAPP-A and free beta-hCG levels are low but detection rate using second-trimester analytes is poor. 6. Although screening protocol has a 90 % pickup rate of aneuploidy, it is not diagnostic. Hence, suspicious screening results will tell of an increased risk of a problem, but not diagnose the problem as being present or being absent. Therefore, a negative screening result may falsely reassure many women who are carrying an affected fetus. Conversely, a false positive result may culminate in termination of a normal pregnancy. The main aim of screening test is to identify a group of women at significantly high-risk of having an affected child to justify the offer of a diagnostic test. 3. Non-invasive screening using fetal blood cells and fetal DNA. (a)Fetal blood cells: The fetal and maternal circulations are separated by the placental membranes, but this barrier is incomplete to cellular trafficking. Bi-directionality and bimodality (cell and cell-free DNA) of fetomaternal trafficking is an established fact. Fetal nucleated cells such as trophoblasts, erythrocytes and white blood cells are found in maternal blood, and have been widely pursued as potential substrates for noninvasive prenatal diagnosis. However, concentration of these cells in maternal blood is very low (1fetal cell in 103-108 maternal cells) and therefore isolation of these cells for cytogenetic analysis requires expensive euipments and great expertise. Isolation of erythroblasts has attracted most attention because they are abundant in early fetal blood, they are extremely rare in normal adult blood and their half-life in adult blood is only about 30 days. Relatively specific monoclonal anibodies against these cells are also available. Trophoblastic cells and white cells are not used for prenatal screening because trophoblastic cells are cleared by maternal lungs whereas half life of the white cells is very long which may lead to contamination from previous pregnancies. A combination of sophisticated physical and immunological methods are used for the identification, isolation and enrichment of different types of fetal blood cells from maternal blood obtained at 12-16 weeks of gestation. Physical methods include triple density gradient centrifugation and micromanipulation techniques while immunological methods include the use of magnetically labelled or fluorescent monoclonal antibodies such as anti-CD71 (transfferin receptor). Commonly used immunological methods are, magnetic cell sorting (MACS) or fluorescence activated cell sorting (FACS). By these methods the fetal cells can be enriched to about 1 in 10-100 materanl cells. Enriched cells are used for the detection of specific chromosomes by fluorescent in situ hybridization (FISH) and for the analysis of fetal genetic loci by single cell PCR. FISH involves the hybridization of DNA probes representing a specific chromosome or chromosomal region to target DNA such as metaphase chromosomes or interphase nuclei. Fetal trisomy is suspected if some of the enriched cells show three-signal nuclei rather than the normal two. Over the past decade, progress has been made towards the isolation and analysis of fetal blood cells from maternal blood, using various enrichment strategies and analysis by fluorescent in situ hybridization with choromosome-specific probes and PCR. It is now possible to identify simultaneously all major chromosomal abnormalities by the use of multicolor probes directed against chromosomes 21,18,13,Y and X in interphase nuclei. Another method that is currently being explored involves culturing fetal blood cells. The sensitivity for detecting aneuploidy using fetal cells is competitive with first or second-trimester maternal serum screening with the advantage that the invasive testing rate may be as low as 0% rather than 5%. But the current technology precludes application of this concept for mass population. However, fetal cells might be analyzed only after a positive serum or ultrasound finding. If fetal aneuploid cells are not recovered, an invasive diagnostic procedure could be avoided. (b) Fetal DNA Isolation of fetal cells from the cellular fraction of maternal blood needs expensive and advance equipment because of the rarity of such cells. Recently, there has been much interest in the use of the non-cellular portion of blood, namely plasma, for molecular analysis diagnosis. Fetal DNA has been identified in the plasma of pregnant women at concentrations much higher than those present in the cellular fraction. Until recently, it was assumed that fetal DNA detected in maternal plasma was cell free and a definitive diagnosis of fetal chromosomal aneuploidies was not thought possible. Studies now indicate that some fetal DNA originates from intact fetal cells. Trophoblasts may be the predominant cell population involved in the liberation of fetal DNA into the cell-free fraction. It was found that the absolute concentration of fetal DNA in maternal serum increases with gestational age, with a sharp rise near the end of the pregnancy. Fetal DNA is cleared rapidly from maternal blood after delivery with a half-life of minutes. Increase in fetal DNA in maternal plasma has been noticed in pregnancies affected by fetal trisomy, and in premature labour, and preeclampsia. Using fetal DNA in maternal plasma for the screening of fetal chromosome aneuploidies is possible, especially in conjugation with other established serum markers. The median fetal DNA concentration in Down's syndrome cases was found to be 1.7 times higher than in controls. Second-trimester cell-free fetal DNA estimation gave a 21% detection rate at a 5% false-positive rate. When added to quadruple marker screening, fetal DNA estimation modestly increased the screening performance above what is currently available in the second-trimester. The relative ease and reliability with which fetal DNA can be detected have thus opened new possibilities for non-invasive prenatal diagnosis. Invasive diagnostic procedures 1.Chorionic villus sampling CVS is a prenatal test that involves taking a sample of some of the placental tissue. Main complications of CVS include severe transverse limb defects and oromandibular-limb hypogenesis [103]. Possible mechanism of severe transverse limb abnormalities is hypoperfusion, embolization and release of vasoactive substances due to trauma during the procedure. There is strong association between the severity of the defect and the gestation at sampling. Decreasing risk and a trend from proximal to distal limb damage with increasing gestational age at CVS provide biologic plausibility for a true association with limb reduction defects [104,105]. CVS as early as 8 weeks of gestation may lead to amputation of whole limb while sampling at 10 weeks only affects terminal phalanxes. Thus, to avoid severe limb defects, CVS prior to 11 weeks of gestation should be discouraged. The increase in the miscarriage rate following this procedure is about 0.5% above the background risk for miscarriage, which is 2-3% at 10-12 weeks of pregnancy. Various trials compared CVS in first-trimester and amniocentesis in the second-trimester and concluded that both procedures are equally effective and safe with similar rates of procedure-induced fetal loss at experienced centers. The main advantage of CVS over amniocentesis is that prenatal diagnosis is achieved during the first-trimester, which allows a couple the opportunities to consider their options earlier in the pregnancy in the event of an abnormal result. However, screening for ONTDs cannot be achieved through CVS. The main indication for repeat CVS is mosaicism. 2.Amniocentesis Amniocentesis was first used for prenatal diagnosis in the 1950's and the feasibility of culturing and karyotyping amniotic fluid cells was first demonstrated in the late 1960s. Although the exact risk associated with amniocentesis is controversial, it is also not a completely innocuous procedure and can result in various complications such as spontaneous abortion, premature labour, placental abruption, intrauterine death and neonatal death. Other reported procedure-related complications include increased risk of respiratory distress syndrome and pneumonia in neonates, talipes and dislocation of the hip, postprocedural amniotic fluid leakage of amniotic fluid, vaginal bleeding, chorioamnionitis, amniotic band formation and rare needle puncture of the fetus. Amniocentesis may results in future reproductive complications secondary to sensitization to Rh and other rare antigens such as Kell. Amniocentesis can be performed as early as 11-13 weeks of gestation. But at this stage of pregnancy, the procedure is not only technically difficult to perform, it is also associated with increased incidence of total fetal loss, talipes equinovarus and post-procedural amniotic fluid leakage. Incidence of failed culture is also high in early amniocentesis. Thus, the procedure should not be performed before 13 weeks gestation unless there are special circumstances. Potential advantage associated with amniocentesis includes; 1.The amniotic fluid can be tested to measure the amount of AFP, which is a diagnostic test for detecting ONTDs 2. Possibility of an abnormal chromosome result due to chromosomal mosaicism confined to the placenta is reduced.

Chorionic villous sampling

8 Weeks pregnancy

Rupture of the Uterus

Incidence: About 1:4000, 95% of cases occur in multipara particularly grand multipara. Causes: (A) During pregnancy (I) Spontaneous: 1.Rupture of a uterine scar: e.g. previous C.S. especially upper segment, myomectomy, hysterotomy, uteroplasty or perforation. 2. Abruptio placenta with severe concealed haemorrhage. Anterior sacculation in case of incarcerated retroverted gravid uterus or posterior sacculation due to previous ventrofixation of the uterus. 4. Rupture of a rudimentary horn at the 4th - 5th month. 5. Perforating vesicular mole. (II) Traumatic 1. Perforation during vaginal evacuation. 2. External trauma. (B) During labour: (I) Spontaneous: 1. Obstructed labour. 2. Rupture of a uterine scar. 3.Grand multipara: due to degeneration and overthinning of the uterine muscles. (II) Traumatic : 1. Internal version: particularly after drainage of liquor. 2. Manual separation of the placenta. 3. Destructive operations. 4.Extending cervical tear due to e.g. forceps or ventose applications before full cervical dilatation. (III) Improper use of oxytocins. Weak uterine scar may be a result to: 1. Imperfect suture with improper coaptation of the edges. 2.Bad haemostasis results in blood clot formation which prevents good coaptation and predisposes to wound infection. 3. Wound infection. 4. Subsequent implantation of the placenta over it. 5. Upper segment caesarean section scar is weaker than lower segment scar. 6. Repeated vaginal deliveries after a previous C.S weaken the scar . 7. Types: 1.Complete : involving the whole uterine wall including the peritoneum. 2. Incomplete: not involving the peritoneal coat. Sites: It depends upon the cause of rupture. (1) In obstructed labour: - It is usually in lower uterine segment. - Usually oblique or transverse. - More on the left side due to; i) dextrorotation of the uterus. ii) left occipito-positions are more common. - Extended tear may pass laterally injuring the uterine vessels leading to broad ligament haematoma formation. This rupture may involve the ureter or bladder. (2)In rupture scar: At the site of the scar. Clinical Picture: (A) Impending rupture : before actual rupture the following manifestations may be detected: 1- Lower abdominal pain. 2- Tender uterine scar. 3- Vaginal spotting (minimal bleeding). (B) Actual rupture: i) Symptoms: 1.Sudden severe abdominal pain : It is differentiated from labour pain being continuous . 2.If the patient was in labour there is cessation of uterine contractions. 3.Shoulder pain on lying down due to irritation of the phrenic nerve by accumulating blood under the diaphragm. 4.Silent rupture: minimal symptoms may occur in rupture lower segment scar due to presence of fibrosis and minimal internal haemorrhage. ii) Signs 1- General examination: Variable degrees of collapse is present according to amount of blood loss. This may appear postpartum in case of traumatic rupture uterus. 2- Abdominal examination: - Scar of the previous operation. - Foetal parts are prominent and felt easy. - The presenting part recedes upwards. - Abnormal foetal attitude and lie. - FHS usually not heard. - The uterus is felt separated from the foetus . - In incomplete rupture, the foetus still inside the uterus with suprapubic painful tender swelling which is an accumulated blood in the vesico-uterine pouch. 3- Vaginal examination: - The presenting part recedes upwards. - Vaginal bleeding may be present. - Contracted pelvis may be detected. - A cervical tear may be found extending to the lower uterine segment and a broad ligament haematoma may be present. Differential Diagnosis: 1. Abruptio placentae. 2. Disturbed advanced extrauterine pregnancy. 3. Other causes of acute abdomen. Management: (A) Prophylactic: 1.Early detection of causes of obstructed labour as contracted pelvis and malpresentations. 2. Proper use of oxytocins. 3. Version is not done if liquor amnii is drained. 4.Forceps application and breech extraction should not be done before full cervical dilatation. 5.Elective caesarean section for susceptible scars for rupture as upper segment C.S. 6.Exploration of the genital tract after difficult or instrumental delivery. (B) Curative: 1- Blood transfusion and antishock measures. 2- Immediate laparotomy. 3- Deliver the foetus and placenta. 4- Explore the rupture site: - If it is amenable for repair and the patient did not complete her family ® repair is done. - If it is not amenable for repair® hysterectomy. Subtotal hysterectomy is less time consuming so it is done if there is no cervical tear. 5- Exploration of the other viscera mainly the bladder. 6- Internal iliac artery ligation may be needed in case of broad ligament haematoma as the uterine artery is usually retracted and difficult to be identified. 7- Vaginal repair: may be amenable if there is slight extension of a cervical tear with accessible apex. Complications: (A) Maternal: 1- Shock. 2- Haemorrhage. 3- Paralytic ileus. 4- Bladder, ureter or visceral injuries. 5- Infection. (B) Foetal :Death due to asphyxia from detachment of the placenta.

Obstructed Labour

Definition: It is arrest of vaginal delivery of the foetus due to mechanical obstruction. Aetiology: (I) Maternal causes: 1-Bony obstruction : e.g. - Contracted pelvis. - Tumours of pelvic bones. 2-Soft tissue obstruction: i) Uterus: - Impacted subserous pedunculated fibroid. - Constriction ring opposite the neck of the foetus. ii) Cervix: cervical dystocia. iii) Vagina: - Septa. - Stenosis. - Tumours. iv) Ovaries : Impacted ovarian tumours. (II) Foetal causes: 1- Malpresentations and malpositions : e.g. - Persistent occipito- posterior and deep transverse arrest, - Persistent mento-posterior and transverse arrest of the face presentation. - Brow, - Shoulder, - Impacted frank breech. 2- Large sized foetus ( macrosomia). 3- Congenital anomalies : e.g. - Hydrocephalus. - Foetal ascitis. - Foetal tumours. 4- Locked and conjoined twins. Diagnosis: It is the clinical picture of obstructed labour with impending rupture uterus (excessive uterine contraction and retraction). (A) History: of - prolonged labour, - frequent and strong uterine contractions, - rupture membranes. (B) General examination : shows signs of maternal distress as: - exhaustion, - high temperature (³ 38oC), - rapid pulse, - signs of dehydration : dry tongue and cracked lips. (C) Abdominal examination: 1- The uterus : - is hard and tender, - frequent strong uterine contractions with no relaxation in between (tetanic contractions). - rising retraction ring is seen and felt as an oblique groove across the abdomen. 2- The foetus : - foetal parts cannot be felt easily. - FHS are absent or show foetal distress due to interference with the utero-placental blood flow. (D) Vaginal examination: 1- Vulva: is oedematous. 2- Vagina : is dry and hot. 3- Cervix: is fully or partially dilated, oedematous and hanging. 4- The membranes : are ruptured. 5- The presenting part: is high and not engaged or impacted in the pelvis. If it is the head it shows excessive moulding and large caput. 6- The cause of obstruction can be detected. (E) Differential diagnosis: 1- Constriction ring. 2- Full bladder. 3- Fundal myoma. Complications: (I) Maternal : 1- Maternal distress and ketoacidosis. 2- Rupture uterus. 3- Necrotic vesico -vaginal fistula. 4- Infections as chorioamnionitis and puerperal sepsis. 5- Postpartum haemorrhage due to injuries or uterine atony. (II) Foetal: 1- Asphyxia. 2- Intracranial haemorrhage from excessive moulding. 3- Birth injuries. 4- Infections. Management: (A) Preventive measures: Careful observation , proper assessment, early detection and management of the causes of obstruction. (B) Curative measures: Caesarean section is the safest method even if the baby is dead as labour must be immediately terminated and any manipulations may lead to rupture uterus.

Obstetric Terms

Presentation: The part of the foetus related to the pelvic brim and first felt during vaginal examination. The presentation may be: (a) Cephalic (96%): i) Vertex: when the head is flexed. ii) Face: when the head is extended. iii) Brow: when it is midway between flexion and extension. (b) Breech (3.5%). (c) Shoulder (0.5%). Cephalic presentation is the commonest as this makes the foetus more adapted to the pyriform-shaped uterus with the larger buttock in the wider fundus and the smaller head in the narrower lower part of the uterus. Position: The relation of the foetal back to the right or left side of the mother and whether it is directed anteriorly or posteriorly. The denominator: is a bony landmark on the presenting part used to denote the position. In vertex it is the occiput. In face it is the mentum (chin). In breech it is the sacrum. In shoulder it is the scapula. Occipito-anterior positions are more common than occipito - posterior positions because in occipito - anterior positions the concavity of the anterior aspect of the foetus due to its flexion fits with the convexity of the vertebral column of the mother due to its lumbar lordosis. * In each presentation, except the shoulder , there are 8 positions. In vertex presentation they are: - Left occipito -anterior (LOA) 60%. - Right occipito-anterior (ROA) 20%. - Right occipito - posterior (ROP) 15%. - Left occipito-posterior (LOP)5%. - Left occipito-transverse (LOT). - Right occipito - transverse (ROT). - Direct occipito -anterior (DOA). - Direct occipito - posterior (DOP). LOA is more common than ROA, and ROP is more common than LOP as in LOA and ROP the head enters the pelvis in the right oblique diameter which is more favourable than the left oblique because: i) anatomically, the right oblique is slightly longer than the left, ii) the pelvic colon reduces the length of the left oblique. Lie: It is the relation between the long axis of the foetus and that of the mother. - Longitudinal in cephalic and breech presentations. - Transverse or oblique in shoulder presentation. Attitude: The relation of foetal parts to each other. - Flexion in the majority of cases. - Extension in face presentation. Synclitism: The posture in which the 2 parietal bones are at the same level. Asynclitism: - The posture in which one parietal bone is at a lower level than the other due to lateral inclination of the head. - Asynclitism is beneficial in bringing the shorter subparietal supraparietal diameter (9 cm) to enter the pelvis instead of the longer biparietal (9.5 cm). - Slight degree of asynclitism may occur in normal labour. (1) Anterior parietal bone presentation: - The anterior parietal bone is lower and the sagittal suture is near to the promontory. - It occurs more in multigravidas due to laxity of the abdominal wall. - It occurs also in contracted flat pelvis. (2) Posterior parietal bone presentation: - The posterior parietal bone is lower and the sagittal suture is near to the symphysis. - It occurs more in the primigravidas due to tense abdominal wall. Anterior parietal bone presentation is more favarouble because; 1. The head lies more in the direction of the axis of the pelvic inlet. During correction of asynclitism, the head meets only the resistance of the sacral promontory while in posterior parietal bone presentation the head meets the resistance of the whole length of the symphysis pubis. 2. In posterior parietal bone presentation the head stretches the anterior wall of the lower uterine segment with liability to rupture. 3. Engagement: - It is the passage of the widest transverse diameter of the presenting part, which is the biparietal in vertex presentation, through the pelvic inlet. - The engaged head cannot be easily grasped by the first pelvic grip, but it can be palpated by the second pelvic grip. - Rule of fifths: 2/5 or less of the foetal head is felt abdominally above the symphysis pubis. - Vaginally : the vertex is felt vaginally at or below the level of ischial spines. - Stations: -Station 0 the vertex at the level of ischial spines. -Stations -1,-2 and -3 represents 1,2 and 3 cm respectively above the level of ischial spines. -Stations +1, +2 and +3 represents 1,2 and 3 cm respectively below the level of ischial spines. - In the primigravidas, engagement of the head occurs in the last 3-4 weeks of pregnancy due to the tonicity of the abdominal and uterine muscles. - In the multipara, the head is usually engaged at the onset of labour or even at the beginning of the second stage due to less tonicity. Causes of non-engagement: (I) Faults in the passenger: 1- Large head. 2- Hydrocephalus. 3- Occipito-posterior positions. 4- Malpresentations. 5- Multiple pregnancy. 6- Placenta praevia. 7- Short cord. 8- Polyhydramnios. (II) Faults in the passages: 1- Contracted pelvis. 2- Pelvic tumours. 3- Full bladder or rectum. (III) Faults in the power: Atony of the abdominal muscles.

Ovulation induction

Introduction: Ovulation induction includes medical induction by ovulatory agents and surgical induction. Ovulatory agents were introduced in 1960, expanded considerably in the last years. Each drug has specific indication, mechanism of action & clinical monitoring. Surgical induction of ovualtion is resorted to when the medical methods of induction fail. It entails making a port through which the ovum is released in cases of polycystic ovarian disease ( PCOD). Pre-therapy work up: 1- Document anovulation (BBT, endometrial biopsy) 2- Rule out and treat reversible pathology (e.g. pituitary tumor). 3- Rule out ovarian failure ( FSH > 40mIU/ml). 4- Ensure that other elements of fertility are intact (good male and tubal factor). 5- Document the couple’s psychological readiness for - Prolonged therapy - Possible failure - Method risk Indication of ovulation induction: 1- Anovulatory infertility. 2- Timing of ovulation for artificial insemination-husband (AIH), artificial insemination- donor (AID). 3- Oocyte maturation for IVF, ICSI,……… Clomiphene Citrate Pharmacology: -Is an orally active non-steroidal compound which is structurally related to ( di-ethyl-stilbosterol (DES). -Exhibits both estrogenic and anti-estrogenic effects. Mode of action: 1- Central action ( hypothalamic): -It occupies estrogen receptors in the hypothalamus (because of its structural similarity to estrogen). -Its dissociation from chromatin is impaired and remains in the nucleus for a longer period blocking the interaction of receptors with estrogen. So, it causes a state of estrogen insensitivity of the target cells i.e. blinding them to the endogenous estrogen level ® inhibition of uptake of estrogen into the hypothalamus and pituitary. ® ¯ –ve feed back effect of estrogen ® ­ GnRH and gonadotropin release ® stimulate follicular activity. 2- Peripheral action ( ovarian): Clomiphene may also act by 2 mechanisms on the ovary; • Direct stimulating effect. • ­ ovarian sensitivity to gonadotropins. Selection of patients: 1- Should have intact hypothalamo – pituitary- ovarian axis. • Intact hypothalamus: capable of producing GnRH. • Intact pituitary gland: capable to respond to GnRH. • Ovary with certain degree of follicular activity (i.e endogenous estrogen present). 2- Should have normal prolactin value. 3- Should have good liver function. So, Clomiphene is used to treat patients with group II WHO which include; 1- Hypothalamic –pituitary dysfunction. 2- PCOD. Clomiphene citrate is ineffective in: 1- Severe hypothalamo-pituitary failure (Group I WHO) 2- Hypo-estrogenic patient. Success of Clomiphene depend on: 1- Sufficient stage of follicular maturation. 2- Body wt. not < 80% of the ideal body weight. Regimens of Clomiphene: [1]. Conventional regimen -Begin by 50mg/day (one tablet) beginning in the 5th day of a spontaneous or progesterone-induced bleeding for 5 days. -However, Clomiphene can be started in 2nd, 3rd, 4th or 5th day of onset of menses. The outcome in terms of ovulation rate, luteal phase defect or pregnancy rate is optimum for day 5. -The pre-ovulatory LH surge is expected to occur 5-10 days after the last tablet (average 7 days) and couples are advised to have intercourse on alternate days for 1w starting 5day after the last Clomiphene tablet. Monitoring of therapy: to document ovulation (a) FSH, LH on day 5 and 9 (b) Estradiol on day 14 (c) Progesterone on day 23 ( > 3ng/ml) However these assays are expensive and time consuming (d) Cervical mucus score and postcoital test (PCT) on day 14 (score 8 or more with ­BBT indicate ovulation). (e) Ultrasound folliculometry daily from day 9 until ovulation (18 – 22mm followed by collapse and fluid in cul-de-sac). (f) Timed endometrial biopsy (document ovulation if endometrial response appropriate). (g) Basal body temperature (BBT); thermogenic shift. -If ovulation is not documented and pregnancy does not occur ® continue another cycle after increasing the dose by 50mg (maximum 250mg/day) till ovulation. -If ovulation documented and pregnancy does not occur ® continue the same dose for other cycle(s). - If menstrual bleeding does not occur within 4 weeks after the last clomiphene tablet and the cycle was ovulatory, a pelvic examination or pregnancy test is done. - Another treatment is withheld if overstimulation is noted (ovarian cyst). - Once an ovulatory dose is reached, treatment should be continued on a regular basis until: 1- Conception occur 2- Other infertility factors are discovered which would prevent preg. 3- Serious side effects occurred 4- The couple wishes to discontinue therapy NB. However, because only 5% of all couples conceive after 6months of therapy, empiric use of other modalities should be considered at that time. - Another treatment cycle begins when: 1- Return of BBT to normal. 2- Intensity and duration of last menses is normal. 3- No signs and symptoms of ovarian enlargement. 4- Rule out preg. by HCG assay. Of all patients who conceive during therapy, 75% of them will do so within the first 3 ovulatory cycles. [2] Incremental or extended regimen: -For patients who did not respond to conventional C.C. therapy larger doses of Clomiphene are used; 50 mg for 5days then increase dose every 5 days by 50mg but maximum dose never exceed 250mg/day. -The treatment period of more than 3weeks does not improve the result. During therapy, monitoring of E2 and U/S until the largest diameter reaches 18-19mm when HCG is injected. [3] Combined Clomiphene therapy: - Used in cases of Clomiphene failure. - The combinations include the following: (A). Clomiphene + human chorionic gonadotropin (HCG): Patients who fail to ovulate with CC alone may ovulate after injection of HCG 5-7 days after CC. Idea: - 80% of anovulatory patients respond to cc by folliculogenesis, endogenous LH peak and ovulation. - 20% fail to ovulate by demonstration of persistent follicles by means of U/S but will do so after 35hr. after injection of HCG 10,000 I.U. -When 100mg Clomiphene fail to induce ovulation, inject 5000-10,000 IU 7 days after the last dose of cc this is followed by another injection 5 days later to stimulate the function of corpus luteum (CL). -Proper timing best with U/S folliculometry is important because HCG when given before follicular maturation favor atresia and inhibition of ovulation. A dose of 5000 I.U is usually sufficient and larger dose increase the risk of hyper-stimulation and multiple pregnancy. -The couple is advised to have intercourse in the same day of injection and in the following 2 days. (B) Clomiphene and HMG: (sequential fashion) -Clomiphene increase the reactivity of the ovary to HMG. This reduce the dose and duration of treatment. The dose of HMG is decreased by 50%. Begin by CC 50mg – 200mg/day for 5 days followed by HMG usually starting with 2 ampoules daily then the dose depend on urinary and serum E2. (C) Clomiphene + GnRH: 1- 100mg CC on days 5-9 plus, GnRH 0.6 – 1.2mg three times daily as nasal drops from day 11-14. 2- GnRH 25 mg 3 times daily on days 5-7 then 100mg CC on days 10-14. 3- GnRH 100 mg daily for 4weeks, followed by CC. (D) Clomiphene + Estrogen: -Estrogen increases the quantity and quality of cervical mucus. • Ethinyl estradiole 20-40 mg for 5 days after the last CC pill. • Estradiol benzoate 1mg for 7 days after 100mg cc • Sequential preparation: estrogen – progestogen; [2mg Estradiol valerate for 21 days & 50 mg Levonorgestrel for 10 days] gives some favorable results. (E) Clomiphene and Corticosteroids: -If there is androgen excess (androgen profile should be obtained), CC is effective if androgen excess is ovarian in origin, but if androgen excess is of adrenal or adrenal/ovarian origin, glucocorticoid should be given. 1- Continuos use: Dexamethzone 0.5mg started 2 weeks before inducing menses and continued while giving Clomiphene and until pregnancy test becomes positive. 2- Interval use: is better than continuos use CC on days 5-9 & dexamethazone on days 5-14 -The effect of glucocorticoid is attributed to its reduction of androgen of adrenal origin and consequently enhances the pituitary gonadotorpin release and ovarian follicle reactivity. (F) Clomiphene and Bromocriptine: -The suppression of prolactin increases the responsiveness of the hypothalamo-pituitary- ovarian axis to CC. -It may be due to direct effect on the ovary or ­ secretion of GnRH and gonadtropin. N.B.: Obese patients has high level of endogenous estrogen (E1) and ¯ SHBG so needs larger dose of Clomiphene to compete with estrogen for hypothalamic receptors. The anti-fecundity effect of Clomiphene: [= disparity between ovulation and pregnancy] There is discrepancy between ovulation rate and pregnancy rate & this is due to the anti-estrogenic effect of CC which interfere with conception by: 1- Impeding ascent of sperm through the cx mucus by altering its quantity. 2- Inhibition of implantation of the blastocyst by interfering with the proper development of the endometrium. 3- Impairing implantation by direct action on fertilized ovum 4- It causes luteal phase defect by • Inadequate stimulation of the endomet. during luteal phase • Direct negative effect of the ovary • Improper development of the ovary Also Clomiphene may cause luteinized unruptured follicle. Results of CC therapy: 1- Ovulation rate 70% 2- Pregnancy rate 40% 3- Multiple pregnancy 8% 4- Congenital malformations are not increased. 5- Abortion rate high 30-40% due to CL insufficiency & antiestrogenic effect on the endometrium. Clomiphene citrate treatment in ovulatory women: • IVF • GIFT • IUI Ovulation is initiated with CC then followed by HMG or FSH then HCG Side effects of Clomiphene: (not dose related and occur with 50mg dose) 1- Vasomotor flushes. 2- Abdominal pain or distention. 3- Breast discomfort. 4- Nausea and vomiting. 5- Visual spots or flashes. 6- Ovarian hyperstimulation syndrome (OHSS): -There is mild but significant ovarian enlargement (14%). - Moderate & severe OHSS is rare. *If the patient is symptomatic, pelvic examination, intercourse and undue exercise should be avoided because the enlarged ovaries are very fragile *Ovarian enlargement regresses rapidly and rarely delays subsequent treatment cycle. 7- Multiple pregnancy (4-9%). 8- Luteal phase defect (LPD): - Due to direct effect on the ovary & endometrium. Also it may be caused by non-physiologic circulation of gonadotropins & E2. - Treatment by: • increasing dose of CC (i.e. CC causes LPD & is used in its treatment). • HCG & progesterone. 9- Abortion (30-40%); due to LPD. 10- Dysmucorrhea: CC fails to produce pre-ovulatory cervical mucus peak despite increased estrogen, due to increased sensitivity of the endocervical glands to the anti-estrogenic effect of CC. N.B.: There is no evidence that the effect of clomid is long lasting & the response is limited to that cycle in which the drug is used. Cyclofenil (Ondogyn) - Chemically related to CC. and is similar to CC in biologic activity. - It is weak estrogenic activity (1/1000). Mode of action: as CC (i.e. acts on hypothalamo – pituitary axis & on ovary). Indications: as Clomiphene Regimen: • 200mg 3 times daily for 5 starting on day 3 or 5 • It may be combined with HMG. And may be combined with bromocriptine Results: ovulation and pregnancy rates are less than with CC: • Ovulation rate 50% • Less multiple pregnancy. • Pregnancy rate 20%. Tamoxifen (Nolvadex) Non steroidal anti-estrogen, structurally related to CC. Advantages over CC: 1- Lack of anti-estrogenic side effects on cervical mucus. 2- Lack of hyper-stimulation syndrome. 3- Lower cost. Dose: 10mg/day for 5 days may ­ up to 40mg/day Side effects: No toxicity but hot flushes are more with tamoxifen. Human Menopausal Gonadotropins -HMG are extracted from urine of postmenopausal women. -It has been in clinical use since 30 years. -It contains FSH and LH (75 or 150 IU of each) • HMG is available in 3 preparations FSH: LH with 1:1 ratio as in pregonal, Humegon FSH: LH with 3:1 ratio as in normegon Pure FSH as in metrodin This pure FSH contains negligible amount (< 1 IU) of LH and was initially developed as a product that might be used in conditions of LH excess such as polycystic ovaries. • A new preparation of genetically engineered recombinant FSH ( r FSH) is now available which has the advantage of being free from any extraneous proteins which are invariably present in most urinary extracted compounds Principles of gonadotropin therapy FSH LH Responsible for recruitment, selection growth and ripening of the dominant follicle • Final maturation of G. follicle • Ovulation • Formation of C.L Indications: (selection of patients): 1- Patients with hypothalamo-pituitary failure (WHO group I) 2- Patients with hypothalamo-pituitary dysfunction (WHO group II) who failed to conceive with C.C. Regimen -No fixed dose -HMG is given by I.M. injection and purified FSH by S.C injection . -The patient is started on day 2 of the cycle with 150 m daily and seen on cycle day 8 and every 1-3 days thereafter for monitoring. -Daily dose depend upon ovarian response. Monitoring of therapy Is done by: 1- Pelvic U/S for the size and no of follicles 2- Estrogen level: • Urinary and blood levels • Effect on cervical mucus The aim of monitoring 1- Assess the effective dose that causes ovarian response 2- Assess the length of time required for follicular maturation 3- Assess the appropriate time for induction with HCG 4- To ¯ the possibility of multiple pregnancy and ovarian hyperstimulation On the day of satisfactory follicular growth (Diameter 18-19mm) and acceptable E2 level ® HCG is given 5000 – 10,000 IU and the patient is instructed to have intercourse in the same day of injection and in the following 2 days The cycle is cancelled and the HCG is withheld and the couple advised to abstain from intercourse if: 1- More than 3 dominant follicles are present 2- E2 level exceeded 2000ng/ml In order to avoid multiple pregnancy and hyperstimulation syndrome. N.B.: Group II: more sensitive to HMG than group I ® more cancelled cycles. Less response to HMG than group I. Results: • Ovulation rate 90% • Preg. rate 50-70% preg. is less in group II due to age factor (advanced age) premature luteinization) • Multiple preg 10-30% • Abortion rate 20% Side effects 1- Hyperstimulation syndrome 2- Multiple preg. 3- Local reaction at the injection site perhaps due to prot. content (patients are switched to purified preparation FSH). Combination HMG therapy [1] Clomiphene citrate + HMG CC 100mg from day 2-6 for follicular recruitment then followed by HMG daily or on alternate days. This ¯ the amount of HMG need However, this regimen is only of use for anovulatory patients of group II who have endogenous gonadotropins and hence endogenous E2 [2] GnRH agonist and HMG combination Principle • Group I patients (hypogonadotropic) have better response and outcome than group II patients (normogonadotropic) • Patients with elevated levels of LH as in PCO are particularly prone to lower success rate after HMG and higher abortion rate this led to the suggestion that converting group II patients into a hypogonadotropic state may improve the results. This is done by giving Gn-Rh agonist before starting HMG, the so called medical menopause, induced by down regulation caused by the agonist. Regimen Complete down regulation (serum estradiol < 50 pgm/ml) takes about 3w if started in the follicular phase and 10-14 days if started in midluteal phase as flare up is accentuated by the natural rise in FSH if the agonist is given in the follicular phase, where the action of the agonist is prolonged. Following downregulation, HMG is started in the usual way but patients may need higher doses with higher costs N.B.: Down regulation may persist for some time after stoppage of the agonist. So, luteal phase support should be given to replace the ¯ed endogenous LH we can give: • HCG 2000 u twice in the 3rd and 6th day following ovulating dose • Progesterone I.M. (50-100mg daily) • Progesterone supp. (100-200mg daily) for 14 days HCG has the Advantage of less frequent administration but it has the disadvantage of - ­ risk of hyperstimulation - Give false +ve preg. test [3] HMG co-treatment with growth hormone This co-treatment with GH ­ serum and follicular fluid IGF, level when compared with standard HMG. Regimen without GH. Although this regimen ¯ amount of HMG needed, it does not appear to improve the clinical response in normal women the ultimate role for cotreatment may be in women who have previously responded very poorly to standard HMG regimen Gonadotropin – Releasing hormone GnRH is a 10 aa protein synthesized and released from the arcuate nucleus of the hypothalamus and released in discrete secretory bursts into the portal circulation and acts to release both LH and FSH . When delivered in pulses, exogenous GnRH acts in physiologic manner to stimulate gonadotropin release. In contrast, when delivered in a continuous fashion, GnRH ttt results in downregulation of pit. Gonadotropin release and suppression of gonadal function Indications: Used in patients lacking endogenous GTH but having intact pit. gland . 1- Hypothalamic amenorrhea 2- Hypothalamic infertility 3- Also used in cases of resistant PCOD Regimen Pulsatile GnRH is delivered by means of small infusion pump that can be programmed to deliver small bolus (1-20 mg) every 60-90 minutes . It can be delivered either subcutaneously or I.V. however the I.V. route of administration appears to be more effective Ovulation usually occurs after 14 days of ttt (10-21 days) when the leading follicle is 14mm or over, the women is instructed to use the urinary LH kit and when the surge is detected, the couple is advised to have intercourse on the following 3 days. Luteal support is needed and done either by: • Continuing GnRH • or using HCG • or Progesterone Advantages 1- More physiologic and safer then GTH 2- Used alone or with cc or HMG 3- Less OHSS. 4- Less multiple preg. Results • Group I : 30% conception rate/cycle and 5% multiple preg. • Group II: lower preg. rate Side effect 1- Formation of anti GnRH antibodies 2- Allergic reaction 3- Infection at catheter site 4- Pump failure Bromocriptine 2 bromo a ergocriptine is a semisynthetic product derived from the family of ergot alkaloids . Indications 1) Anovulation in cases of : • Pit. microadenoma • Hypothyroidism ( ­TSH ) • Following steroid contraceptives • Following drugs • Following electroconvulsive therapy 2) Inhibition of postpartum lactation 3) Acromegaly 4) Parkinsonism Action : ¯ RRL (dopaminergic on hypoth – direct on pit) Dose : 2.5mg twice daily for 21 days with building of the dose Side effects 1- Nausea and dyspepsia 2- Constipation 3- Postural hypotension 4- Nasal congestion 5- Muscle cramps Superovulation: Induction of multiple ovulation in patients who are ovulatory Used in: - Long unexplained infertility with IUI - Assisted reproductive technology ART (IVF – GIFT) * Unexplained infertility Induction of ovulation is used as an empirical therapy either alone or with IUI CC ® 3-5% preg. rate/ cycle And higher with IUI combination HMG ® 18-15% preg. rate/cycle * IVF – GIFT Superovulation is used as preg. rate is related to the no. of embryos replaced Surgical induction of ovulation [1] Multiple ovarian puncture: ovarian drilling: Laparoscopic multiple ovarian puncture (15-20 sites) using cautery diathermy or laser vaporization lead to a response similar to that with HMG in CC resistant PCO patient Also, patients who don’t ovulate spontaneously following the procedure become more responsive to medical therapy and up to 60% of them will ovulate when cc is recommended The advantage of ovarian puncture over HMG are 1- One step ttt 2- No monitoring is required 3- The risk of iatrogenic multiple preg. is eliminated Disadvantages 1- Operative procedure with general anaethesia 2- Postoperative adhesions: however it is less than ov. wedge resection which is now obsolete Mechanism: removing part of hormone producing ovarian tissue ® ¯ androgen and inhibin levels ® ¯ FSH, LH ¯ and resumption of spont. ovulation The choice between ovarian puncture and HMG in cc – resistant patients will depend on: 1- Local availability of facilities 2- Experience 3- Patent wishes [2] Ovarian wedge resection: Removing part of the ovary now obsolete as it ® post op. Adhesions