IVF & Genetic Center Jabalpur Madhya Pradesh, India

Dear Colleagues

Hello

In this issue we are discussing two very important issues ,one is complication of IVF and other is prevention of cancer cervix.

Multiple embryo transfer is a very tricky issue and it poses great dilemma for the IVF physician. In India as there is no government funding for the IVF treatment ,the IVF centers are at great stress as their name is at stake with all IVF attempts. Patients spend their own money and they wish for good results. There is a effort to achieve pregnancy at all cost with each attempt. For that we use more drugs to get more eggs and eventually to get more embryos. We transfer at least three good embryos and sometimes even more, to get a pregnancy. Some times this creates a peculiar problem of multiple pregnancy. More than two fetuses is risky as there is fear of abortions and premature delivery ,which is a great stress to the couple , psychologically and monetarily.

Blastocyst transfer of one or two embryos is solution , otherwise Multifetal pregnancy reduction is the only choice, which has its own complications.

Human Papilloma Virus is one of the main causative factors in carcinoma cervix in female . Pap smear is one of the screening method to prevent it ,but with low speficity and sensitivity. HPV detection ( onchogenic variety ) helps us to make good decision making.

Our first national training on IVF and Embryology was fantastic. Participants enjoyed it to a great extent.


With best wishes
Dr.D’Pankar Banerji

Embryo reduction in triplet pregnancy, achieved by IVF from oocytes collected from Pouch of Douglas—Case Report

This patient is Kaushal aged 35 yrs. We are following her since three issues. As it was confirmed by the sonography that all the three embryos are implanted,it was a matter of concern as triplet pregnancy will be very hogh risk for her and her babies.

She had been a case where, out of three oocytes ,two oocytes were released before egg collection at the time oocyte pick up. The fluid collected in Pouch of Douglas was aspirated and our embryologist Dr.Mrs. Rinku found two oocyte in them. All of them were placed in dish and convetional IVF was done. All of them fertilized and they were in sequential culture .First in fertilization mediaum for one day ,them in cleavage media for two days and them in blastocyst media for two days. And day five transfer done of all the three blastocyst .One was of good quality (see figure below )



After 14 days ,pregnancy test was done and she was found to be positive.

After 10 days sonography was done and it was evident that all the embryos implanted and three sacs were visible.

Embryo reduction was planned at 9 weeks of pregnancy.

Patient was taken in operation room (empty stomach ) and in lithotomy position Inj.propofol was admininstered as anesthetic agent by our anesthetist.

Transvaginal USG done to confirm the alive fetuses . Needle guide applied on the Tranvaginal probe . One fetus distant from the probe was found to be smaller than the others and targeted for Injection. Injection KCl ( 2 meqv/ml) 2ml loaded in two ml syringe.A cyst aspiration needle from Cook ( 17 G)attached to the syringe and air in the needle is displaced by KCl.

Smaller fetus was then placed in the track of the needle by manuovering the probe.It was a difficult option to inject the distant fetus ,as we have to avoid the other sacs and have to traverse the large amount of uterine tissue.

The needle tip is pushed to the heart of the fetus and KCl 1.5 ml is injected in to the heart directly. There is asystole within few seconds. Needle is placed for a while and color flow was started to confirm the asystole.

It is very important to do this injection in one shot otherwise inadvertent spill of KCl will create more harm and reinjection is always risky and there is great chance of abortion.

After confirming the asystole ,needle is removed and again color dopler is applied to see the other fetuses and to confirm their viability.

Next day again the check sonography and color Doppler done to confirm the asystole of the injected fetus and it was found that other fetuses were not harmed.

( It is very important to do the check sonography, some times the fetus recovers the asystole and it is very dangerous to have a KCl injected live fetus.



Our experience in Multi Fetal Pregancy reduction at Ideal Fertility

Total four cases :

1st : Triplet pregnancy ,one reduced , Patient delivered at 37 weeks by caesarian section .Both the babies are alive and healthy

2nd : Quadruplet pregnancy ,two fetus injected and still carrying the pregnancy ,two alive fetuses at 38 weeks.

3rd : Triplet pregnancy , one injected . Patient aborted after one month of the procedure ( it has been reported by the other authors that 9-15 % of the pregnancies may undergo miscarriage after 4-8 weeks of injection.).

4th : The present case , still the pregnancy is going well.

Conclusion : Multifetal pregnancy reduction is a tricky affair and it psychologically demanding for both , the couple and the gynecologist. We are trying to reduce the number of embryos transferred after IVF and mostly going for one or two ,good quality blastocyst transfer

DNA PCR for Human Papilloma virus from chronic cervical erosion, A way of prevention of Cervical Carcinoma—Protocol followed at Ideal Fertility

Cancer of the uterine cervix the most common malignant tumour in the women in world-wide and represent a major public health problem in south east asia. Human papilloma virus ( HPV) has emerged as a major pathogen associated with this disease.HPV are members of papova virus family and contain a double stranded circular DNA genome with a typical size of about 7900 bp.

It has been shown from several studies that Human papilloma Virus infection is a good marker for women with cervical neoplasia and pre-cancerous lesion. Women persistently infected with certain “onchogenic “ HPV types ( type 16 and 18 )show a high rate of progression of dysplasia to invasive cancer of the cervix.

The diagnosis of HPV infection may facilitate early identification of women at increased risk of developing cervical cancer. Pap screening program has been the mode of cervical screening for several decades . The biggest limitation of the Pap test is the poor sensitivity (50-80%) and the need for frequent repetition.

Sample collection protocol for HPV testing

• Cervical brush : Collect cervical cells from the exocervix and endocervical canal using the Cervical sampling brush. Rinse the brush thoroughly in the transport tube containing sample collection buffer for the laboratory.
• Tissues : Tissue biopsy in an HPV collection tube with collection buffer. Outside samples are collected frozen.

What affects the test ?

• The use of douches ,tampons and vaginal creams or vaginal medications within 24 hours before the procedure may interfere with the test results.
• An insufficient cervical cell samples or menstrual bleeding may interfere with the test results.

More recently , the Polymerase chain reaction  method is being used to rapidly and accurately detect Human Papilloma Virus in clinical samples.
This method is reporting a sensitivity and specificity of greater than 90 % and is proving to be useful.

Steps Of PCR :

• Samples are received at Lab : The samples may be , Cervical sample or tissue.
• Samples are treated prior to extract the DNA of HPV. Steps are different for different samples. Mostly they are centrifuged at high speed to get a pellet which are rich in tissues or cells infected by HPV.
• Extraction HPV DNA : Here various extraction kits are used which are commercially available . Cell proteins are lysed with Protinease K (enzyme). This reaction takes at higher temperature .DNA of HPV dissolved and precipitated with alcohol (95-100 %) . This precipitated DNA is isolated and dissolved in nuclease free water of TE buffer
• There are three steps in actual PCR .These are denaturing ,annealing and extension. During denaturing the double stranded DNA of HPV. is separated in single strand at 92 to 95 deg.Cel. for 30 sec to one minute. Then one specific part of DNA is targeted and primers are applied in the presence of T.que polymerase. This step is called annealing and usually done at the temp.58-62 deg.cel.for one minute Taq is an enzyme that helps to extend the synthesis of copy of particular segment in the presence of nucleotides and this is at temp 72 deg. This gives a new copy of particular and specific segment of DNA of the HPV called extension.
• This cycle is repeated number of times app. 20 to 30 times in a instrument called thermal cycler.
• If the samples contain a Human Papilloma Virus then only the particular DNA of virus will be amplified and its presence will definitely confirm , that the person is infected by HPV.
• Amplified DNA is invisible to naked eye and has to be stained by a fluorescent dye.
• Amplified DNA is then poured in small well made on agarose gel and put on in very mid electric field and left for electrophoresis.
• Amplified DNA moves according to its weight and compared with controlled samples , negative and positive and markers .
• After one or two hours of electrophoresis the agar plate is placed in fluorescent illuminator and bands of different DNA are visualized . If sample DNA correspond with the positive control then diagnosis is positive and if with negative then it is negative .

First national training program on IVF and Embryology conducted successfully

At ideal fertility ,First training program was conducted successfully. It was an intense hands on schedule for the participants for Module I and II and conducted from 17th june to 19th June 2007. We have decide to take only two participants per batch so that the personal one to one communication was at its best.

Two participants were Dr. Nitish Biswas from Kolkata and Dr.Shyamala from Hyderabad. Both were practicing gynecologist. Two IVF cases were planned during the training schedule so that the participant had a live feeling of the actual procedure. The participants were in the operating room and in culture room during the procedure. They were trained to make culture media for IUI . They retrieved the oocytes from mammalian ovaries and learnt ,how to place culture in a culture dish. Fundamentals of embryology and lab procedure were covered by our embryologist ,Dr.Mrs. Rinku Banerji.

There was one to one discussion and presentation in the field of reproductive endocrinology and various induction protocols for IUI and IVF. The induction protocols and oocyte pickup and embryo transfer part was covered by Dr.D’Pankar Banerji.

Feed backs from the participants :

Dr.Biswas –“ many many thanks for the training. The embryology and post training advice is very much needed when we start our own.”

Dr.Shyamalla – “your training was very educative and friendly. we got an idea about IVF. Please try to interact after we leave the training and guide us further”.

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