Reproductive Genetic Diagnostics

Reproductive Generic Techniques
BY DR G M WANI Ph.D,DMVet
Dr Paras wani MD COMMUNITY MEDICINE
Dr Gazanfer WANI MBBS
Dr TALIT ZAHIR BUMS

Introduction

DNA: Is deoxyribose nucleic acid. This small cellular portion contains all information required to synthesize cellular and extra-cellular structure. A DNA is a double strand structure. Each strand of DNA consists of nucleotide units which are arranged in a linear polymer. The units are connected with pentose sugar at 5 carbon unit to a phosphate group and via its 1 carbon to one of the four bases Guaine, cytosine adenine, and thymine. The bases are arranged by the principles of Watson-Crick Base-Pairing Systems where Guanine base has purine base type and has 2 rings 3 hydrogen bond and base pair designated as G-C cytosine base has pyrimidine base type 3 hydrogen bonds and G-C base pair Adenine has purine base type 2 rings and hydrogen bonds but base pair is A-T which is similar to thymine who has pyrimidine as base type, one ring and 2 hydrogen bond. With this simples description of DNA readers are expected to read some fundamental molecular biology book to understand this chapter fully in its further elucidations. One copy of the entire double-stranded DNA content is referred to as haploid genome in humans. It has thus 3 x 109 base pairs in 23 separate molecules. Each cell is diploid with two copies of this genome in 46 chromosomes (22 pairs of autosomes and one pair of sex chromosomes) thus, every DNA strand or chromosome has a capacity to store enormous information both in autosomes and sex chromosome units.

Besides this vast information DNA contains scaffold chromatin which packages DNA with histones. The DNA not used by cell in template remains in an merit state tightly packaged within the chromatin scaffold of the chromosome. Thus, the usual function of any cell can be utilized to enfold this vast information. Once we understand and use this chromatin scaffold, a new generation of human and mammalian genetic techniques shall come into being. The cure, repairing diseases and will be easy ailment at genomic level. A new understanding of genetic applications are thus in offering in future. This chapter shall endeavor to propose a few known techniques in use at present in mammals.

Genetics Reproduction

New techniques such as in vitro fertilization, IVF, pre-implantation, genetic diagnosis (PGD) and prenatal diagnosis shall be our focus in this section. The medical reproductive genetics techniques presently available can be enumerated as ;

a) Maternal Serum Screening: This technique called (BUN) is useful in detecting fetus likely to be effected with down syndrome. A serum test between 10-14 weeks of gestation helps to detect the disease. BUN study involves the measurement PAPP-A (Pregnancy Associated Plasma Protein) PAPP-A and free B-HCG (Human Chrorionic gonadotrophins with muchal translucently thickness.
b) Genetic Analysis of Fetal Cells: This technique allows to differentiate between fetal and maternal cells during pregnancy and helps to isolate single fetal cells for genetic manipulation or disease cure. Chromosome analysis can also be made on these fetal cells.
c) Aminocentesis (EATA): Easily removal of amniotic fluid between 13-16 weeks of pregnancy helps to diagnosis of various cell type and diagnosis of disease of neonate.
d) Many other techniques like maternal serum markers, in utero magnetic resonance imaging, preimplantation genetic diagnosis are being performed at present. The result obtained by such reproduction genetic techniques in the past shall be discussed.
e) Stem cell, cloning and ART assisted reproductive techniques are also in use with ethical restriction in humans
f) Research on Epigenetic process is also initiated
g) Use of genetic techniques for predetermining the capacity and capabilities of primodial germ cell containing oocyte shall be focus of future genetic engineering experts. While we shall review the earlier 7 set of technique in use at present. The last postulations shall help young genetic to unveil the potentialities of oocyte.

1. Material Serum Screening

Material Serum Screening is a blood test (MSS). This helps to determine certain abnormalities in the fetus. It is done in the first part of pregnancy. Generally it is done during first trimester of pregnancy in women. However, it can be also done in 2nd trimester of the pregnancy too. The following syndromes can be detected by this test. (1) Down Syndrome

It is also known as trisomy 21, because of the baby in women caries 3 copies of chromosome 21 instead of usual two. Downs Syndrome is associated with defects like intellectual impairment, heart abnormalities etc.

2. Edwards Syndrome

Also known as trisomy 18. it occurs when fetus has three copies of chromosomes 18 instead of usual 2. Defects include neural deformity of hands and feet and intellectual impairment.

Neural Tube Defects

These are detected during 2nd trimester. Neural tube defects result in failure of the brain and spinal cord eg. Spina bifida, i.e. incomplete bifurcation of spinal vertebrae. Spinal cord and tube protudes through the gap instead growing down into the abdominal organs and legs leading to paralysis of the legs.

These are preliminary tests more precise and accurate diagnostic test to diagnose these syndromes are provided by Amniocentesis or Chorionic villi screening material serum screening can be coupled with nuchal translucency detected at ultrasonography around 12 weeks of pregnancy, where pocket of fluid at the back of neck of the fetus can be detected.

The best judge to advise patients is her doctor. However, these tests indicate risk factors and can not be taken as definitive. This has necessitated genetic counseling. The modern technique of prenatal genetic counseling and evaluation has become important to rescue mothers of the mental agony. Should we be able to detect these chromosomal aberrations in infancy or at "oocyte or sperm" stage, much break through could be made to reduce the ensuing handicap babies. Is it possible let us review.

3. Genetic Analysis of Fetal Cells

Following fetal cell genetic analysis can be undertaken as one date;

(a) Prenatal diagnosis of sick cell anemia
(b) Quantitative analysis of fetal DNA in maternal plasma
(c) Male fetal progenitor cells, persist in maternal blood

An non-invasive aneuploidy detection technique has been described (Bianchi, Dev 1997) under the title progress in genetic analysis of fetal cells circulating in maternal blood. The technique, its diagnostic value and other aspects has been current. Opin.Obstel.Gynaecoal 1997, April 9 (2):121-5. thus as on date the technique are a decade old and have been reviewed and its advanced version too are available in literature (www.ncbi.nlm.nih.gov; www.linkingtube.elsevier.com). A precise documentation of the prenatal diagnosis of spinal muscular atrophy in human foetus has been published on web. Beroud (2003. linkingtube.elsevier.com). similarly reports and publication on sickle cell anemia Ceieng, MC, 1996 www.nature.com. Many PCR based non-invasive techniques have been described. New methods of magnetic cells sorting too are in vogue Skizaqa, A 1999; Content Karger Corn, gives more details. Spinal muscle atrophy diagnosis was made at genetic analysis of fetal cells. Saker, 2006 (doi-wiley.com). Gender diagnosis can also be made at examination of foetal cells (www.fags.com). Many books on www.google.com. describes fetal cell and fetal DNA detection technique in Maternal Blood.

Prenatal diagnosis of diseases in fetus has entered a new era of advancement with the advent of intact fetal cell analysis along with fetal nucleic acids freed from fetal cells. All these end products carried in maternal plasma or serum have revolutionized the genetic disease diagnosis in fetus. The availability of circulating fetal cells in the mothers blood gives new diagnostic challenges to the researchers. Precise technique to isolate fetal cells from the mothers bloods are lacking, however, efforts are one and many NIH awards and fellows are at work to come up with a refined diagnostic technique. The earliest signal indicate increased bidirectional movement of fetal and maternal cells and nucleic acids in abnormal pregnancies. This bidirectional trafficking as it called is an significant sign which should alarm an clinician that some thing is wrong and be may plan proper diagnostic advise or test for this patients. This type of cells shall help in the diagnosis of such defects as preclampsia and fetal aneuploidy. The evidence of fetal stem cells and progenitor cells presence in the blood of mother shall be of double significance in future. It shall help to cure many ailments even when pregnancy terminates or culminates. Does these fetal progenitor or stem cell have effect on maternal instinct commonly recognized.

Types of Cells Entering maternal Circulation

Fetal erythrocytes and platelets enter maternal blood stream during pregnancy. Decidual material of trophoblast derived placental masses or cells were also detected in maternal blood. As back as 1969. The Walknowski test had the distinction of detecting 46 x 4 Karyotype by analysis maternal blood of 30 pregnant women.

The Walkanowki Karyotype experiment were published when primitive cell culture and analytic techniques were available. With the modern Fluorescent Chromosome banding techniques are available. Trophoblast sprouts are unique diagnostic material in maternal blood for diagnosis of genetic make up and fetal disease patterns in future. What is needed more intensive research on fetal biology which remains a neglected fluid of study, although USAD and other funding agencies in USA have given it priority I. human placentation is of unique order which results in the loss of mothers spiral arteries and increases their diameter. Thus, the material exchanges and the presence of trophoblast remnants in maternal blood may be of special interest in future.

Fetal Viability

Some of fetal biological viability tests described which included (a) Nuchal-thickness scan, isolation of fetal cells from maternal blood serum or blood circulation as a whole. Triple test serum biochemistry are know techniques. These techniques are on blood of mother and do not carry much risk, the use of amniocentesis, cordocentesis, fetal skin biopsy, coaelomic fluid aspiration and chorion villous manipulation and needs to be done strictly on the advise of doctor. Practioners and doctors should make it a point o discuss and explore all possible ways for accuracy to minimize errors and omissions.

Many websites give details of fetal viability charts. Many patient views can also be seen on www.gogle.com .

Amniocentesis

Amniocentesis is removing small amount of fluid from amniotic sac. It is fought with risks of membrane rupture or pregnancy loss. Ameniocenthesis is preformed during ultrasound examination. A pocket of amniotic fluid is pin pointed by ultasonography first. Then a needle is guided by a trained and licensed medico through the abdomen into the pocket. Some fluid is removed. Fluid is sent for cell culture. They are examined for chromosomal abnormalities if any. There are risks of miscarriages in this technique.

Faster methods of chromosome analysis are now available. Fluorescent in situ hybridization technique is one such method.

It uses special dyes targeting specific chromosomes. Each dye glows a different color shine, thereby differentiating the chromosome. Normal cells as already explained process of 23 chromosome pairs, this dye of fluorescence can detect only 4-5 different chromosomes. This technique is commonly known as FISH (Flourescena in situ Hybridization). With this limitation of number FISH picks up potential chromosome portion having additional or missing chromosome. FISH involves chromosome animally screening. FISH has advantage of quick results within few hours and not days as is routine with culture methods.

Another potential risk with amniocentesis is it time of accuracy. It is performed around 2nd trimester of pregnancy. Perhaps it is too long for a decision.

The other alternative method at preset is CVS (Chorionic Villus Sampling). Its results are available earlier than the amniocentesis. We can conduct the test during 1st trimester of pregnancy, this technique is also an ultrasonography guided catheter insertion via cervix into developing placenta. A small biopsy needle is used. The material removed by suction. The material is subjected to cell culture or FISH examination as described. Because CVS involves minisurgical or injection procedures via cervix it has been seen that it carries higher miscarriage risks. The special expertise to locate villus and a direct manipulation of fetus and mother writes off the little advantage of easily diagnosis. These techniques have not been experimented in other mammals like Bovine, Ovine or Caprine. This is an open filed for reproductive geneticists and theriogenologists.

Firstly to minimize the efforts of amniocentesis and CVS fluid collection process. Secondly perhaps more studies on the fetal development biology are needed. Trophoblast do leave their impressions on the uterine caruncles in ruminants could that material be of any potential genetic diagnostic value needs research.

Another potential research fluid open is for pharmacologists and immunologists. The uterine injection, loss of fluid through needle aspirations are common sequel to miscarriages following amniocentesis. We have the limitation of antibiotic use in-utero. Perhaps safe drugs could be invented using animal primate and small ruminant models. A variety of options have been suggested by us in our earlier book on Animal Agriculture Biotechnology Embryo Biotechnology and Various papers (Wani, 1996, 2008,2009). Alpha-Fetoprotein values in amniotic fluid obtained during early amniocentesis 11-13 weeks of pregnancy, revealed that although aminocenthesis reference points 11,12 or 13 weeks stand established, yet they have limitations and thereby the clinical applications risk

1. Stem – Cell Cloning and ART

Many reviews and books are available on stem-cell and nano-cell technology and its potential use in health and disease of mammals. A broad review has been given earlier. Here we shall discuss its potential benefits or harms to public health in general.

Recent review and investigations on pharmacogenetics show personalized effects of drugs on individuals. Inventing a individualistic prescription as on today appear impossible. However, it there is a hope to do so. We know genetic make up has a response drug or in other words drug targeting at genome level explains the personalized Medicare. Once we unveil the genetic make up of a patient perhaps our drug therapy could be genetically motivated and equally more effective. This is where stem-cell research will lead us in future. On 25th sept. 2005 Royal Society of UK professes "Medicines personalized to patients still decade away" thus on 20th sept. 2005 we hope to have a break through in this field.

It will need more and more reproductive pharmacologists to venture gene targeting and come up with new therapies. Prof Ian Wilmut, the father of cloning "Dolly the Sheep" a Cambridge Veterinarian has already been promised therapeutic cloning license on 8th Feb. 2005.

Half a century ago Watson and Crick described the structure of DNA, today in 2009 we are concerned to use DNA driven methodologies for disease diagnosis and cure. Drug targeting gene has been the subject of many review, today we profess drugs so minute to target "DNA" bases for repair of human misery and sufferings.

Many reviews on treatment of muscular dystrophy using stem cells has been published (Mackenzie et al. 2001) wound healing in mice Sylvester et al. 2000 and use of mesenchymal stem cell graft and its differentiation in ovine uterus (Liechty et al, 2000). Bone marrow transplantation and colonal detection (Kim, et al. 1999) have been some of the pioneering publications on the topic. Multileanage expression of stem cells give a promising prospect of stem cell transplants replacing organ transplants. It may even cure defects in developing fetus by regenerating a new set of known chromosome map after mincing the available defective pair. These are future projections of stem cell research envisioned after study of works of Flake and Zanjani 1998, Leahey et al. 1998 and Porada et al. 1998.

Assisted Reproductive Technology (ART)

Methods to achieve pregnancy by artificial means is termed as Assisted Reproductive Technology (ART). Many manuals such as in vitro fertilization OCR, AZH, ICA, ZIFI and PGD, Gift, SSR are available on websites to give details on medication, commonly known as fertility medication. Many new expansion to ART are now reported like Transvaginal ovum retrieval (OCR). This technique consists of inserting a small needle through the back of vagina. It is a ultra sound guided insertion. It guides needle to ovarian follicles. The fluid is collected and retrieved back for examination of oocyte. This is in fact an oocyte collection method operative in vivo.

This technique need more perfection in small ruminants where laparoscopic or manually guided ovarian aspiration poses difficulties. Many such methods were in operation in sheep and goats (Wani 1981, 82), Goel 2001 in CIRG has already attempted transcervical oocyte collection in goats. These are many reports on transcervical embryo delivery and collection procedures known in Bovine and Ovine practice (Wani 2009).

Now many other assisted techniques are in operation like Assisted Zona Hatching(AZH). This techniques pieces a hole in the zone for hatching of the blastocysts science of reproductive techniques have been deve4loping at a greater pace. Only in 1982 while micromanipulation caprine and ovine embryos Cambridge groups of "Dolly the Sheep" fame were in limelight because of in vitro culture of micro manipulated embryo. The Willadson technique of encapsulating micro embryo in empty zona-free eggs shells was very time consuming. A hours would pass on and one has not been able to free a zona from its nucleus. The muco-gel culture to seal off penetrated zona was yet another comersome process of 1981. son later in 1982, zona-free blastomeles were cultured which paved new way to present zona-hatching in utero and many other techniques. Like intracytoplamsic sperm injection (ICSI), autologous endometrial co-culture, zygote intra fallopian transfer, (ZIFT), many new methods and techniques in assisted reproduction are published. Reviewed and discussed, both in animals and women. (Wani 1996, Nowshahrim 1993, Wani and Wani 2008, 2001, Wani 2006, 2007, 2008, 2009).

The technological advancement has given dozens of hitter to unknown technologies to the world of reproductive science, such as GIFT, Gamete Intrafallopian Transfer, PGD- Preimplantation Genetic Diagnosis, FISH-Fluorescent in suit hybridization.

Many others like sex selection and sperm sorting has been successfully applied in women. Artifical insemination, surgical sperm retrievel (SSR) or epididymal sperm retrievel (ESR) have been discussed and tried with success (Wani and Wani 2003) Wani 2008, Wikipedia.org) Frozen Embryo Transfer (FET) has soon to be replaced with in-vitro-foetus transfer or whole-foetus-transfer IFT or WFT whatever the name.

The use of these techniques is associated with risks, as such a "Warning" for their use. No attempt be made by reader as a novice to these techniques. Only registered medical practitioners licensed to do so, may attempt to use these techniques in humans or animal. Some critical points of there use can be summed up as;

a) Increased risk of fetal defects have been reported 1,2,3
b) Major defects in 2% cases were observed

Naturally conceived have deformities of the order of 4.4%. however, maternal age do effect these rates even in normally conceived pregnancies. Genetic disorders, DNA damage, gene expression low birth weight, visual impairment, cerebral palsy are some of the defects reported using ART.

Membrane damage with increased expression of the membrane fusion protein NAPA and Annexin A3 were reported.

The above narration refers to the published work of Mackenzie et al. 2001, on stem cells, Sytvester et al. 2000, on wound repairs and other workers Kim et al. 1999, Van Vooris, 2007 on clinical practices of assisted reproductive techniques. These review entads various works of Kurinczuk et al, 2004 on in vitro-fertiliztion birth defects besides the published investigation of Zhang et al. 2009, and more recent publication on cerebal palsy, developmental delay and assisted reproductive techniques reported by Hvidtijarm et al. 2009. Many reference and statements refer to the publications in mother earth news. Com and Jain et al, publications on insurance published in new England Journal of Medicine init slates volumes.

Maxkenzie, T.C, Shaaaban, AF, Radu A, Flake A.W 2001
treatment of muscular dystrophy by in-utero stem cell transplant surgical forum L11:328-329

Sytvesterm K.G; Nesbit, M, Radu, A Hertyn, M; Adzick, C.N and Crombelholme, T.M 2000
Adnevoviral-mediated gene transfer in wound healing; Acute inflammatory response inhuman skin in the SCID moue model. Wound repair and regeneration 8:36-44

Kim, HB, Shaaban, A.F; Milner, R; Fichterm C; Flake, AW, 1999.
In utero bone marrow transplantation induce tolerance by combination of clonal detection and energy. J. Pediatr.surg. 34(5) :726-729

Zanjanim ED; Almeida, Poroda, G, Livingston, AG, Flake, AW, Ogawa, M. 1998
Human bone marrow

CD 34 cell engraft invivo can undergo multilineage expression including giving rise to CD 34 + cells. Experimental Hematology 26: 353-360
Assisted Reproductive Technology Wikipedia.org.

Van Vooris, B.J, 2007
Clinical practice IVF
New England Medicine Journal 356 (4) : 379-386

Kurinczuk, J.J, Hensen, M, Bower, C. 2004
The risk of birth defectws in children born after assisted reproductive technologies
Curr.opin.obstel.gynaecl 16(3) 201-209

Also CK, Keppler, N.K.M, Romitti, PA, Budellier, W.T, Ryan, G, Sparks, AE, Van Voothis, B.J 2005
Invitro fertilization is association with an increase in major birth defects
Fertility sterility 84 (5): 1308-1315

Zhang, Y; Zhang, YL, Feng, C; 2008
Comperative proteomic analysis of human placenta derived from assisted reproductive technology. Proteomics 8:4344

Hvidtijarm, D, Schender, D Jacobson, B. Savaerke, C Thorsen, P. 2009
Cerebal palsy, autism spectrum disorders and developmental delay in children born after assisted reproductive conception. Systemic review and meta analysis. Arch pediatr. Adolesc.Med 16 (3) : 72-83

Motherearthnews.com. the sperm crisis

Jain, J; Harlow, BL, Homstein, Md 2008
Insurance coverage and outcome of invitro fertilization. New England Journal of Medicine 347 (9) : 661-666
Infertility treatments NHS direct online.