APPLICATION OF VITRIFICATION IN GENETIC ART EHICHIOYA JOY U. CLINICAL EMBRYOLOGIST NORDICA FERTILITY CENTER, LAGOS. AFRH SEPTEMBER 2017.

OVERVIEW INTRODUCTION GENETIC ART WHY GENETIC SCREENING PGD/PGS VITRIFICATION PRINCIPLE OF VITRIFICATION STAGES OF VITRIFICATION OTHER INDICATIONS FOR VITRIFICATION FACTORS TO CONSIDER FOR SUCCESSFUL VITRIFICATION CONCLUSION

INTRODUCTION Assisted reproductive techniques is constantly evolving. Over a decade ago, in vitro fertilization has been revolutionized by different techniques such as vitrification as well as the introduction of genetic tests.

Early 90’s; evolution of genetic diagnosis. An evolution that had a lot to do with the development of reliable Vitrification Methods. The results of IVF cycles have improved significantly, allowing the detection of genetic anomalies and enabling the selection of embryos without aneuploidies.

GENETIC ASISSTED REPRODUCTIVE TECHNIQUE Treating infertility among couples ART Treatment of human infertility. Genetic screening. new reproductive techniques

WHY GENETIC SCREENING Chromosome aneuploidy (abnormal number of chromosomes) is a major cause of IVF failure. Most embryos with aneuploidy do not implant, and those that do often miscarry during the first trimester of pregnancy.  Using preimplantation genetic screening to selectively implant the most viable embryos can help; Increase pregnancy and live birth rates. Reduce miscarriage rate. Select euploid blastocysts to support single embryo transfer (reduce mutliples) Decrease risk of abnormal offspring

GENETIC SCREENING: FOR WHOM? Advanced age (>35) have a higher risk of having babies with a chromosome problem such as Down’s syndrome T21, Edward’s syndrome T18. Recurrent IVF failure (2/3 or more) Family History of a known genetic disorder. Recurrent miscarriage (Normal Karyotype) Repetitive implantation failure Severe male factor and sperms known to be at high risk of having chromosome problems such as DNA fragmentation (breakage of sperm DNA into pieces). Abnormal sperm aneuploidy test (SAT).

HOW? PGD/PGS Pre-implantation Genetic Diagnosis /Pre-implantation Genetic Screening is a laboratory procedure performed in conjunction with in vitro fertilization (IVF) to help screen for anomalies in embryos. PGD Selection of genetically normal or carrier embryos from couples with known inherited mutations (nuclear DNA) chromosomally balanced embryos from couples with known chromosomal rearrangements Aim to transfer an embryo that will develop into a healthy child PGS Selection of Euploid (normal chromosome complement) embryo/oocyte. It involves checking the chromosomes of embryos conceived by IVF or ICSI for common abnormalities. Chromosomal abnormalities are a major cause of the failure of embryos to implant, and of miscarriages Aim To improve IVF delivery rate

PROCESS Embryos by IVF Biopsy (Day5 Blastocyst) Cell diagnosis EGG COLLECTION BIOPSY @DAY 5 VITRIFICATION SEND SAMPLE REVIEW OF RESULTS FET Embryos by IVF Biopsy (Day5 Blastocyst) Cell diagnosis

LIMITATIONS TIME for analysis

VITRIFICATION In the last decade, Vitrification has become the standard method for cryopreservation Its appearance have had a huge impact in assisted reproduction techniques, redefining procedures like Embryo Transfer. Because of the improved vitrification methods, selection of fresh embryos for transfer is more and more often replaced by frozen embryo transfer.

Comparisim of slow freezing vs vitrification Vitrification is the glass-like solidification of a solution at a low temperature without ice crystal formation, which is made possible by extreme elevation in viscosity during freezing. This can be achieved by extremely high rates of cooling and/or increasing the concentration of the cryoprotectants . Unlike slow freezing, vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified and outside the cells in the surrounding solution .

PRINCIPLE OF VITRIFICATION Vitrification involves exposure of the cell to high concentration of cryoprotectants for a brief period at room temperature The cells are initially pre-equilibrated in a cryoprotectant solution of lower strength resulting in dehydration of the cell and its permeation with cryoprotectant This is followed by a very short incubation (<30 seconds) in higher concentration of cryoprotectant solution (40%) Rapid plunging into liquid nitrogen.

Cryoprotectants Cryoprotectants are basically antifreeze that we add to the solutions in which the cells are being frozen in to protect them from membrane damage and ice crystal damage. They have a second function of stabilizing the membrane and protecting it from damage during cryopreservation. And thirdly, they provide a "hyper-osmotic" environment that helps the process of dehydration, which draws the water out of the cells.

STAGES OF VITRIFICATION Oocytes Zygote 2 – 8 cell embryo Blastocyst

DEVICES Open ….. Direct contact of embryo with LN2 Cryotop Cryoloop Cryoleaf

Closed ……indirect contact of embryo with LN2

OTHER INDICATIONS FOR VITRIFICATION Freeze- all cycles Oocytes/Embryo pooling (poor responders) High number of embryos Delayed motherhood (social reasons) Patients with ovarian hyper stimulation syndrome (OHSS) Sub – optimal endometrium before embryo transfer. Chemotherapy.

BLASTOCYST IMPLANTATION RATE POST THAW Tseng-Kai Lin et al, 2010 41% Pei-Yun K.U. et al, 2012 43.2% Nordica Unpublished Data (IVF) Nordica Unpublished Data (IVF + PGD) 42.11% 43.48% .

MATERIALS AND METHOD Repro plate, straws, pipettes and media. Equlibration for 15minutes. 10 – 12 minutes for 2pn, 4cell or 8cell 12 – 15 minutes for blastocysts. It is the same procedure for Oocytes and Embryos 1 minute, 30seconds for vitrifying and loading.

LOADING The oocytes/embryo is loaded on the cryotop under the microscope with minimal volume of VS2 The excess VS should be removed by aspirating using pipette. The cryotop is plunged directly into liquid nitrogen and the straw capped.

FACTORS TO CONSIDER FOR SUCCESSFUL VITRIFICATION TIME VOLUME TEMPERATURE

CONCLUSIONS Vitrification is rapidly spreading all over the world and it seems to be ready to replace the slow cooling procedures. Utilizes higher concentrations of cryoprotectant that allow shorter exposure time to the cryoprotectants. It requires loading of embryos in carriers with a small volume of cryoprotectants which provides a significant increase in the cooling rate from 20,000 to 30,000oC/ min. Minimizes osmotic injuries. Very simple protocols. An efficient and consistent vitrification program brings flexibility in genetic IVF as any cycle can be stopped at any time if suboptimal conditions occur. Vitrification and frozen embryo transfer are highly effective for PGD/PGS and deserve wide usage. Hence, the importance of improving the technical proficiency of embryologists as this is also key to achieving success in vitrification.