IVF & Genetic Center Jabalpur Madhya Pradesh, India

Dear Colleagues

Hello

In this issue I am dealing with few interesting cases we came across in our set up along with a scientific topic on genomic imprinting. These days are of stem cell technology and scientist are really puzzled at the mechanism that governs the variable and differential expression of the human genome .

The inactivation of genes during cell differentiation is believed to involve epigenetic modifications of chromatins .Epigenetics is defined as nuclear inheritance that is not based on differences in DNA sequences . It is best exemplified by cells comprising different tissues expressing different proteins while having the same genetic make up. Epigenetics is believed to involve differential DNA methylation, histone acetylation, chromatin configuration as well as other mechanisms. These epigenetic signals are stably transmitted during cell division but are reset in each generation in the gonad during fetal development, therefore epigenetic signals are not inheritable from one generation to the next but stably maintained within the generation.

Three interesting case reports are also mentioned ,Hope you will enjoy reading them. For the interested readers ,if they have missed earlier issues,as we are not able to reach all of our friends due to various constraints , they can get them in archives present at Newsletter section of www.idealfertility.com.

All the best for now

Sincerely yours

Dr.D’Pankar Banerji

Genomic Imprinting

Genomic imprinting is a phenomenon helps to express a preferential gene and suppress other gene ,so that the total dose of protein synthesis is balanced.

To understand this we need to understand some basics of genetics. We get 23 chromosome from our father and 23 from the mother. Means Each chromosomes are in pairs. One pair of chromosome is like two identical looking rows of single storied houses, in front of each other, in a colony. The colony has a particular address in the city ( Nucleus ) and that is the numerical identification of that particular pair, may be 1or 2 up to 23.

Each house in that colony has a particular address or location which cannot be changed physically , and this address is called Locus (pl.Loci ).Hence there are two Loci which are in front of each other ,one maternal other paternal , in two opposite row housing.

Each Locus has its own occupants. These occupants are like tenants which keeps on changing whenever required .These occupants are Alleles . If the occupants are same in both the loci then the person is called homozygous for that particular allele , if the occupants are different then the person is called heterozygous.

If these alleles speak and do some fruitful work for the community (Cell ) then they are called Genes. Their voice is called Genetic expression .

If the alleles do not perform any work and their use is only for the purpose of address identification of the active alleles then they are called markers or Genetic marker.

Hence for each pair of chromosome there are two identical Loci and they may or may not have identical  occupants or alleles at that address.

Genes and Genetic markers are made up of DNA. Genes are useful DNA and markers are Junk DNA ( ?).

Whole amount of DNA of cell is called Genome .

When both the alleles( Genes ) of two identical Loci work together then the output of work may be good if material ( protein ) is in scarcity. But if they produce more than the requirement for the colony (cell ) then there product may create problem and may become garbage .If the cell can not handle that garbage to get rid of ,then it may die  or may misbehave, which might be harmful for the body.

To prevent this ,body by some way (still unknown ),inhibit one allele from functioning so that product generation can be controlled .This inhibition of alleles or genes may be of paternal origin or maternal origin. This selective inhibition is called Genomic Imprinting .

This imprinting behavior of a gene is already decided when embryo forms. A zygotic nucleus has an imprint memory that is retained forever; this controls when genes are expressed. Functional differences between parental chromosomes are heritable, and survive activation of the embryonic genome. Data derived form both pronuclear transplantation experiments and classical genetic experiments indicate that the maternal and paternal genetic contributions to the mammalian zygote nucleus do not function equivalently during subsequent development These observations suggest that there is differential genome imprinting during male and female gametogenesis.

The significance of knowing this behavior helped us to understand :

1. Why one X chromosome is active in female and other remains dormant( because body is happy with only one X chromosome as it happens in males ),Lyon Hypothesis
2. Do we change the behavior of an embryo when we make them outside the body as in IVF.
3. Do we interfere with the imprinting when we do Intra cytoplasmic sperm injection.  Because it has been shown that some diseases are more common in ICSI babies , large size babies because lack of imprinting on one Insulin like Growth factor gene ).
4. Why differentiation of totipotent stem cell to differentiated cells like skin , liver, nerve etc cells do not express the proteins of cells of other lineage.
5. Why chromosomal deletions of same area creates different diseases in male and female
6. Does inhibition of imprinting of genes creates neoplasia or cancer.
The branch of science which deals with the imprinting behavior of body and cells is called Epigenetics .

2.Failed conventional IVF and then rescue ICSI ( Intra-cytoplasmic sperm Injection )--Case report

A patient named R.K. ( 31 yrs. Normal weight )came for IVF. She was a case of poly cystic ovarian disease. Husband’s semen analysis shows good number and quality of sperms. Long protocol ( Inj.Luprofact daily ) was used after a progestagen withdrawal bleeding. Ovulation induction was done with Inj. Fostine ( FSH ) 150 IU for 4 days and then Inj. Menodac ( HMG )150 IU for 5 days. Twenty five oocytes were retrieved after 36 hours of Inj Ovidac ( hCG ) 5000 IU . After 6 hours of maturation of all oocytes ,,insemination was done with around 50,000 sperms per oocyte in microdrops under oil.

After 18-20 hours, fertilization check was done and it was evident that, not a single oocyte was fertilized and there is no attachment of sperms in zona pellucida of any egg. All the eggs were denuded and twenty oocytes have been shown to release their polar bodies ( M II ). Intra-cytoplasmic sperm injection was done with freshly prepared sperms. Next day the fertilization check shows 13 fertilized oocytes, with grade I pronucleus ( PN ) ten ,and grade II PN ,three. Twelve embryos survived well up 8-10 cell stage .Embryo transfer was done on day 3 after ICSI.

Inference : There is always a fear of failed fertilization in conventional IVF. ICSI for all patients is also not practical because of economics and other reasons .But  failed fertilization in conventional IVF in first attempt confirms that couple needs ICSI in their subsequent attempts. Rescue ICSI might be a proposition in selected cases.

3. Robertsonian Translocation –Rare variety of Down Syndrome---- Case report

A lady named V.R. aged 26yrs came to us with female child of 5 month . The child have been referred to us for the confirmation of diagnosis of Down Syndrome by a paediatrician of our city.

Down Syndrome cases are with typical cranio facial features includes palpebral fissures, small and low set ears, a protruding tongue and broad nasal bridge.

In about 85% of the cases,Down syndrome results from the presence of an entire additional chromosome 21 ( nondisjunctional trisomy ). In 3%- 5% of the cases, Down syndrome results from translocation or attachment of  another chomosome to the chromosome number 21,  ( mostly chromosome number 14, )

Here in this case the 22 number chromosme is attached to 21.
And mosaic composition is the cause of only few cases.

Nondisjuctional trisomy is usually sporadic and shows a well defined relationship to maternal age.

Where as those cases involving translocations may be familial and unrelated to maternal age, means the recurrence of another Down child is higher.
When a woman has a history of a child with Down syndrome ,it becomes important to know the type of chromosomal defect found in that particular child because the risk of occurrence in future pregnancy will be depending on the type of defect.

If the child with Down syndrome is of translocation variety ,it is imperative to do the karyotyping of the parents, as one of them might be the carrier

If karyotype of either parent is normal then the defect in the child may be de novo and risk of having another child is 1 %.

If mother’s karyotype is normal but the father has a balanced 13/21,14/21,15/21, or 21/22 translocation then the risk of another affected child will be 2-3 % .

If father’s karyotype is normal and the mother is the carrier of a balanced and the mother is the carrier of balanced 13/21, 14/21, 15/21, or 21/22 translocation the probability of having another affected child is 11.9 % .

If any parent carries a translocation of 21/21 then the recurrence risk will be 100 %.
Any of the parent affected by translocation will appear normal as they have normal amount of genome , but their numerical analysis will show 45 chromosomes as two chromosomes are attached together.

Pregnancy achieved by IVF of oocyte collected from Pouch of Douglas—Case report

As has been published in the previous issue ,We have a patient named Mrs.Kaushal ( age 35 yrs ) for conventional In-vitro fertilization. Her two follicles were ruptured before the oocyte retrieval procedure. Collection at the Pouch was aspirated along with existing follicle and all the three oocytes were recovered on 25th April 2007. All the them fertilized and were cultured upto day5 and three blastocyts were transferred ( one with good quality ).Patient did not received any progesterone support for five days as she left the station for some urgent work.

Pregancy test ( urine card test )was done after 14 days of Embryo transfer ( 13th May 2007) and it was positive.

On 21st May 2007 Transvaginal scan was done and it was found that all the three embryos are implanted and three distinct intrauterine sac visible with good chorionic activity and visible fetal pole and yolk sac.

We did total nine IVF in the month of April 2007 and out of them six showed the urine test positive for pregnancy. We achieved almost 66% pregnancy rate, which we feel was an extraordinary result.

Training in IVF and Embryology

Module I : Ovulation induction and Intra Uterine Insemination ( One day ),Rs.2000
Module II : Conventional IVF and fundamentals of Embryology( Two days ), Rs.20,000
Module III : Intra cytoplasmic sperm injection, Micromanipulation ( Two days ) Rs.50,000
For details contact ,two participants per batch

Dates :
Throughout the year

Drafts should be in favor of Dr.D’Pankar Banerji, payable at Jabalpur.

Stay can be arranged in nearby hotels at an extra cost Rs.250-1000 per day
Lunch will be served during the training session.

Faculty :
Dr.D’Pankar Banerji ,Consulting Gynecologist and Infertility specialist
Dr.Mrs.Rinku Banerji,Embryologist

Certificate will be issued after the course to participants

Sale and purchase of the used equipments

* Ultrasonography machine for schimadzu SDU 350- convex & vaginal probe 2000 make. with probe converter contact +91-9998968631." Dr.kalpana khandheria

Letters to the editor:

I welcome your views and comments in this column and will be published in the forthcoming publications