Future direction of pre-implantation genetic diagnosis (PGD): comparative genomic hybridization (CGH).

Issue 2 : July – September 2010

Figure 1 : CGH principle

Somjate Manipalviratn, M.D. A signifif icant proportion of unsuccessful pregnancy is caused by numerical chromosome imbalance or aneuploidy supported by the evidence that >50% of fif irst trimester abortuses are aneuploidy. FISH was introduced to reveal aneuploidy in embryos in order to improve the pregnancy success. However, whether PGD using FISH technique will improve pregnancy rate of IVF treatment cycle is still controversial due to its limitation in the number of chromosome studied. This is limited by the number of distinctf luorochromes which are available to label the DNA probes. The more probes applied simultaneously the more overlapping signal will occur. Though signal overlapping can be partly overcome by using computerized imaging, signal overlapping of the same flf luorochrome cannot be delineated. A common approach is to use FISH for 5-6 chromosomes simultaneously and then once these results have been analyzed, the signals are washed-off and re-probing the nucleus for other 2-4 chromosomes is done. This cannot be done repeatedly as DNA degenerates and signal effif iciency is reduced with each round of re-probing. Recent evidences have shown that aneuploidies are not limited to particular chromosomes but can occur to any chromosome which can result in pregnancy wastages or infertility so there is an interest in analyzing every chromosome in a single blastomere for PGD. Conventional karyotyping using G-banding technique for the analysis of metaphase chromosomes has been reported but is too ineffif icient at the single cell level to be applicable to PGD. To date, the most successful approach for complete karyotyping of single cells is comparative genomic hybridization (CGH). CGH has originally been applied to the detection of chromosome copy number changes in tumors. CGH does not require the preparation of chromosomes from the sample and in a single experiment reveals the copy number of every chromosome segment larger than 5-10 Mb in size depending on technique used. Originally, published CGH protocols require 0.2–1.0 µµg of sample DNA whereas a single cell contains only 6 pg. This is later overcome by using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify the whole genome before the CGH analysis. CGH works by a comparison of the sample or test DNA with known normal or reference DNA. Test DNA and reference DNA are labeled with a green and red f lfluorochrome respectively and then simultaneously applied to either human male metaphase chromosomes or microarray slide (fiF igure 1). The green:red flflf luorescence ratio would normally be 1:1 for each euploid chromosome. If the test cell is monoploidy for a particular chromosome/segment then there would be a decreased ratio of green:red flflf luorescence in that particular chromosome/segment. If the test cell is polyploidy for a particular chromosome/segment then there would be an increased ratio of green:red flf lfluorescence in that particular chromosome/segment. The advantage of CGH is that besides analyzing the copy number of all chromosomes, it can also reveal chromosome microdeletions and/or microduplications. Microarray-based CGH provides a higher resolution than CGH onto metaphase chromosomes which is limited to about 5 Mb. In order to perform PGD-CGH, the embryos should becultured to blastocyst stage as blastocyst biopsy provides higher number of DNA copies for whole genome amplifif ication compared to the biopsy of cleavage stage embryo. Moreover, the embryo freezing/thawing protocol must be effif icient as all embryos need to be frozen after the biopsy and be transferred in the subsequent frozen/thawed cycle as the PGD-CGH result is generally available after 2-3 weeks from the day of biopsy. In conclusion, CGH offers a new paradigm of pre-implantation genetic diagnosis to allow clinicians to offer aneuploidy testing of all chromosomes in pre-implantation embryos with an expectation of improving IVF success.

An interesting genetic disease: Von Hippel-Lindau disease (VHL)Pannatee Sanpong, M.Sc.

Von Hippel–Lindau disease (VHL) is a rare genetic condition which is inherited in an autosomal dominant pattern (Figure 1), with a frequency of approximately 1 in 36,000 live births. VHL is a hereditary cancer syndrome predisposing to a variety of malignant and benign neoplasms, most frequently retinal, cerebellar, and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma, and pancreatic tumors.

genetic disease. Where a couple are interested in PGD for detecting a single gene disorder, you can refer the patient with the following protocol. The fif irst process of PGD-PCR is a specif ific test kit preparation, at this stage the important information is required for test kit preparation are; Mutation reports of a couple and / or relative family members. Blood samples or DNA sample of a couple and relative family members

The procedure for test kit preparation 1. Clinician with a clinical geneticist or genetic counselor will arrange a consultation to a couple for the information of IVF/PGD-PCR treatment. 2. A couple should be given an interview with a PGD scientist for family history and take family pedigree. The PGD scientist will assess a feasibility of test kit preparation from mutation report and indicate the appropriate blood samples to be collected from family members for test kit preparation. 3. Collect blood samples from the couple and relevant family members. This should either be a) 3-4 ml blood collected in an EDTA tube or, b) blood collected onto a blood card. In some circumstances a buccal sample (mouth swab) may be collected. 4. The test kit preparation will commence when PGD scientist has received all necessary mutation reports and blood samples. The test kit preparation normally takes 8-10 weeks to complete. * The patient should be informed that no stimulation can commence

until a completed test is available at Superior A.R.T.*

5. PGD scientist and nurse coordinator of Superior A.R.T. will report a completed test to clinician and patient. Following this a stimulation cycle can commence. 6. Please notify nurse coordinator at Superior A.R.T. immediately when the stimulation cycle has started, in order to prepare case setting-up. Furthermore, you can directly bring your patient to start stimulation cycle at Superior A.R.T. 7. If a patient comes through for any subsequent test cycles, PGD scientist at Superior A.R.T. should be notif ifified immediately at least 2-3 weeks before or at start of any drug treatment. The scientist will review amount of test kit available. If insuffif icient then a new test kit will have to be made up and tested with no additional cost.

The disease results from a mutation in the Von Hippel-Lindau tumor suppresser gene (VHL gene) which located on the short arm of chromosome 3p25.3. The defect of VHL gene causes the loss of VHL protein activity results in an increased amount of HIF1a, and thus increased levels of angiogenic factors. In turn, this leads to unregulated blood vessel growth which is a predisposing factor for tumor formation leading to the disease.

VHL may be diagnosed when one of its associated symptoms start to cause discomfort in the person suffering from the disease. Retinal and CNS hemangioblastomas are pathogno-monic for the disease and their presence is a critical clue to diagnosis. Surgery is the mainstay of treatment for tumors that arise in patients with Von Hippel-Lindau disease. Current Medication for VHL-related cancers mainly targets the HIF pathway such as VEGF has recently been approved by FDA.

However, VHL is one of the single gene disorders thatSuperior A.R.T. can provide a preimplantation genetic diagnosis using polymerase chain reaction (PGD-PCR) to exclude the embryos affected with the disease. Therefore, only the disease-free embryos will be transferred to the uterus forfurther implantation. Commonly, PGD is used when a couple is aware of the possibility that their offspring will inherit a

Reference: 1. Von Hippel–Lindau syndrome. http://www.ncbi.nlm.nih.gov/omim/193300 2. Von Hippel–Lindau disease. http://en.wikipedia.org/wiki/VHL_disease 3. Von Hippel-Lindau Disease: Treatment & Medication. http://emedicine.medscape.com/article/950063-treatment

Figure 1 illustrate an autosomal dominant inheritancepattern : Whenever one parent carries the autosomaldominant faulty gene copy, there is 50% chance thatthe child will inherit the faulty gene and will thereforebe affected by the disorder.From : http://www.gig.org.uk/images/gen-dominant.gif

Figure 2 illustrate a slit lamp photograph showing retinaldetachment in Von Hippel-Lindau disease.From: http://en.wikipedia.org/wiki/VHL_disease

Mutation reports of a couple and / or relative family Mutation reports of a couple and / or relative family

Advisory Board Prof.Somboon KunathikomDr.Viwat ChinpilasDr.Somjate ManipalviratnMr.Sarayuth AssamakornMs.Pannatee Sanpong

Editor Anin Gujral

It was a truly amazing technology we have just next door! I have a disease called Von Hippel Lindau (VHL), a very rare case of genetic disorder -- my wife and I did not want to pass it on to our children. Although this was the f ifirst attempt with Superior A.R.T., it was our second time that we tried to overcome this with PGD-IVF technology. About fif ive years ago when we worked and lived in the US, we carried out PGD-IVF procedure with the clinic in the US when we found out that this technique could help to screen the mutated genes. Unfortunately, we ended up with only one tested negative for VHL embryo left for transfer – the IVF step was not successful. We decided to leave this behind – we naturally had our f ifirst child a year after that. A few years later when returning to Thailand, during my internet search, I found that Superior A.R.T. can perform VHL screening through their PGD/PCR-IVF procedures. It was not an easy decision this time after our unsuccessful experience that we had. To us, doing this

Abstract : Von Hippel-Lindau (VHL) is a rare autosomal dominant genetic condition involving the abnormal growth of blood vessels result in little knots which are called angiomas or hemangioblastomas in some parts of the body which are particularly rich in blood vessels such as in the cerebellum, spinal cord, kidney and retina. These abnormal vessels usually occur in the retina. As a result, loss of visions is very common. Some of these tumors are benign, while others are cancerous. There is a wide variation in the age of onset of the disease, the organ system affected and the severity of effect. This disease is caused by mutation of the Von Hipple-Lindau tumor suppressor (VHL) gene on the short arm of chromosome 3 (3p25.3). About 80% of cases were inherited this mutation gene from one of their parents. ICSI and Preimplantation Genetic Diagnosis (PGD) with PCR technique is a method to avoid VHL gene transmission from the parents to the child. A 37 years old healthy women and her 37 years old husband who has experienced multiple organ tumors from VHL has come to Superior ART to consult about their plan to have unaffected child(ren) since last year. They have one child, 2 years old, who also has got VHL mutated gene transmitted from his father. They have ever done ICSI and PGD-PCR in United States 3 years before but not succeeded. Development of PGD-PCR kits was carried out 10-12 weeks before ICSI program. Once the test kit was ready, ovarian stimulation was performed using Antagonist protocol. 12 eggs were retrieved with 8 MII. Unfortunately, only one 2PN embryo was developed. This one embryo was cultured to day 5 blastocyst and freeze. Two month later, Short protocol was performed to re-stimulate the eggs. 12 eggs were retrieved with 10 MII and resulted in four 2PN. Biopsy was performed on two fresh and one frozen-thawed hatched blastocysts on day 6 following PGD-PCR analysis. The PGD-PCR results showed that there were 2 unaffected embryos. These two unaffected blastocyst were transferred into uterine cavity under ultrasound guided and result in a singleton pregnancy. The pregnancy is now going on. This is the fif irst case report of successful pregnancy after ICSI and PGD program for VHL for private IVF center in Thailand.

Case reportCase reportPGD-PCR success for VHL disease : A case report fromSuperior A.R.T.

Sharing Patient’s JourneySharing Patient’s Journey

Author :Piyaphan Punyatanasakchai, M.D.

Obstetrician and GynecologistInfertile specialist

kind of procedures is both emotional and physical challenging. However, after consulting with Dr. Piyaphan Punyatanasakchai and Khun Pannatee Sanpong, the genetic scientist at Superior A.R.T., we were confif ident and that this could work and decided to have another try. Thanks for Dr. Piyaphan, Khun Pannatee, and their professional and dedicated staffs at Superior A.R.T., my wife was transferred with two tested VHL-negative embryos and she is now 20-week pregnant. We are expecting our second child late this year. We are excited and can not wait to see how happy our family will be with another new coming member. This will not at all be possible, without faith and technology, and more importantly professional team at Superior A.R.T. By the way the clinic is just fifif ifteen minutes away from where we live - we will certainly bring our children to visit the clinic and the team again soon. This is our current family members

thThe Best SME 2008

14 AustCham ThailandBusiness Awards 2008

Activities

Superior A.R.T. held the seminar in the topic of “PGD-PCR technology and the success story of patients using thistechnology” on the 12 May 2010 for specialists and other doctors who were interested at Bangkok Hospital.

Superior A.R.T. held the seminar in the topic of “PGD-PCR technology and the success story of patients using thistechnology” on the 27 May 2010 for specialists and other doctors who were interested at Bangkok Nursing Home (BNH)Hospital.

Superior A.R.T. was invited by Tu Du Hospital, Ho Chi Minh City, Vietnam to present in “The 10thVietnam-France-Asia-Pacifif ic Conference on Reproductive Health Program” in the topic of “Management of low ovarianresponse to stimulation for IVF”. Dr.Somjate Manipalviratn, our Obstetrician and Gynecologist Reproductive Endocrinologyand Infertility Specialist was a speaker on the 14 May 2010 for specialists and other doctors who were interested.

Services :

IVF/ ICSI In vitro fertilization / intracytoplasmic sperm injection.

TESE/ PESA Testicular sperm extraction / Percutaneous epididymal sperm aspiration.

PGD Preimplantation genetic diagnosis for aneuploidy screening, balanced translocations, single gene disorders and HLA matching.

ET Embryo transfer

Oocyte Vitrifif ication Storage of female eggs for future use

Sperm freezing Storage of male sperm for future use

Embryo freezing Storage of surplus embryo after embryo transfer for future use

Centre for Assisted Reproduction Techology and Preimplantation Genetic Diagnosis Address : Bhakdi Building, 2 / 2 Wireless Road, Lumpinee, Pathumwan, Bangkok 10330 Thailand Tel : 66 (0) 2 255 4848Fax : 66 (0) 2 255 8455E-mail : services@thaisuperiorart.comWebsite : www.thaisuperiorart.com