Visual evaluation of early (~ 4-cell) mammalian embryos
How well does it predict subsequent viability?
Marie-Noël Bruné Rossel
Student Master of Science Degree in Mammalian Reproduction and Fertility
Scottish Agricultural College. University of Aberdeen, Scotland
Tutors: Dr. Tom Mc Evoy, Dr. Maureen Wood
TABLE OF CONTENTS
- Literature review
- Material and methods
This thesis is based on work carried out by myself, unless stated otherwise (in vitro maturation, in vitro fertilisation and in vitro culture until day 2 was carried out by Gilbert McCallum, pyruvate assays were undertaken with the help of Vanessa Moreira and both also helped on Day 7 evaluations in absentia), and has not been accepted in any previous application for a degree. All sources of information have been acknowledged by means of references.
(Creation) Every man may see it; man may behold it afar off. Behold, God is great, and we know him not, neither can the number of his years be searched out.
Science: A little science takes you away from religion; a lot brings you back to it.
Gustave Flaubert ("Le Dictionnaire des idées reçues")
But there remains one final question which, if we could make him look up from his enthralling occupation, we should like to put to this great master of the art of life. In these extraordinary volumes of short and broken, long and learned, logical and contradictory statements, we have heard the very pulse and rhythm of the soul, beating day after day, year after year, through a veil which, as time goes on, fines itself almost of transparency. Here is some one who succeeded in the hazardous enterprise of living; who served his country and lived retired; was landlord, husband, father; entertained kings, loved women, and mused for hours alone over old books. By means of perpetual experiment and observation of the subtlest he achieved at last a miraculous adjustment of all these wayward parts that constitute the human soul. He laid hold of the beauty of the world with all his fingers. He achieved happiness. If he had had to live again, he said, he would have lived the same life over. But, as we watch with absorbed interest the enthralling spectacle of a living soul living openly beneath our eyes, the question frames itself, Is pleasure the end of all? Whence this overwhelming interest in the nature of the soul? Why this overmastering desire to communicate with others? Is the beauty of this world enough, or is there, elsewhere, some explanation of the mystery? To this what answer can there be? There is none. There is only one more question: 'Que sais-je?'
Virginia Woolf, 'Montaigne' (1925)
To my family, Mom, Dad, Pato, Mamina, you taught me the meaning of love, the importance of being different, of searching for challenging changes in life, of how, as Robert K. Hall once said, “Flapping your arms can be flying!”
And to all the outlaws of the mind everywhere, all the crazy spirits that don’t adhere to laws but fight against the dullness of mind while trying to find their own way in the universe.
I would like to thank the constant support of Drs. Tom McEvoy and Maureen Wood for providing me with bibliography, guidance, knowledge and for giving invaluable psychological support. I also want to thank all the help provided by Alex Reis in the statistical analysis and bibliography and Gilbert McCallum in the lab. Thank you all for your patience and help.
I am deeply indebted to the help and constant support of my friend Vanessa Moreira (“the yellow cat”) who has helped me in the lab, allowing me to achieve this work and who, most importantly, opened the wings of her amazing person and friendship to me.
I would also like to thank Margaret King who opened the doors for me to this wonderful country that has become my second home.
I would like to thank many people who changed my life in Scotland. The mango and jasmine of my eye, Valentin, my Scottish family, Jeff and Margaret Brett who gave and taught me love in a Christian way. Alan Campbell and all the Findochty Brethren who helped me through with their constant love and prayers.
I will always be indebted to the amazing people that led me into the reproductive field. At home, Dr. Juan Carlos Scasso, Gerardo Bossano, Lidia Cantu, Jose M. Montes, Guillermo Caprario. In Europe, Prof. Johan Smitz, Prof. Rita Cortvrindt and the invaluable friendship and support provided by Dr. Giovanni Coticchio.
Thank you so much Nicolas Negrin for encouraging me to discover this new world and for always giving the best vibrations, in the north and in the south.
Also all my friends back home, thank you for bringing back a little Uruguay in each email. I cannot thank my family enough, they are the reason of happiness, wild dreams and magic in my life.
A total of 220 bovine embryos from 5 different replicates obtained from abattoir-derived material were assessed on day 2 post-insemination according to the criteria used in a human IVF clinic, the Aberdeen Fertility Centre. Embryos were assigned to independent, randomly selected groups of ten, each group of ten representing a different “patient”. Of these 10 eggs, 5 good or viable eggs were “selected”. The other 5 embryos were termed “unselected”. On day 7, blastocysts were classified blindly (i.e., operator unaware of whether they were “selected” or not) according to an adaptation of the criteria used by Lindner and Wright (1983) for bovine embryo morphology and evaluation. Blastocysts were also measured (diameters, μm), tested for pyruvate metabolism indices and then fixed for cell counts. Preliminary studies (Experiment 1) carried out with two batches of embryos determined the time when most of the bovine embryos would be at the 4-cell stage, in order to ensure that timing of egg “selection” would be appropriate and to account for any differences between human and bovine embryo growth. Embryos in Experiment 1were in three different treatments: SOFaaBSA; SOFaaBSA followed by SOF + 10% serum; and SOFaaBSA followed by SOFaaBSA + 2% lipoprotein solution. Differential growth and viability of embryos assigned to these three treatments was also assessed. Preliminary data showed that the greatest prevalence of 3-4 cell embryos in SOFaaBSA occurred between 44 hours and 56 hours post insemination. The presence of serum increased blastocyst yields. Use of lipoprotein supplement in SOFaaBSA supported blastocyst formation. Unsupplemented SOFaaBSA had the lowest yields of blastocysts. Thus sequential culture proved to be the system that gave the best blastocyst yield. In the subsequent “selection” study (Experiment 2) all 220 eggs were cultured in SOFaaBSA until day 4 and thereafter in SOF+10% serum+vitamin E (100 μM). Final results showed that Day 2 embryos that were selected gave the higher yield of blastocysts and also of grade 1 blastocysts on day 7. Unselected embryos that reached blastocyst stage on day 7 showed no significant difference in cell counts or diameter (171.58 μm ± 2.65 vs 170.42 μm ± 5.41) with the selected group. This showed that even though just a few developed, those unselected embryos that did survive were comparable in quality and viability to selected embryos. These results are discussed in relation to the repercussions such findings, if they were to be echoed in human ART, could have ethically, scientifically and in relation to patients in a human IVF clinic, where embryos are selected for transfer and cryopreservation on day 2 and the unselected ones are discarded.
Assisted reproduction techniques, embryo, blastocyst, cleavage, in vitro fertilisation, pronucleus, embryo metabolism, multinucleation, preimplantation development, embryo morphology.
See Glossary in Appendix 1 for some definitions.
Many diverse factors affect embryo viability. The morphological systems routinely used to grade human embryos can be of limited use as many factors that affect the long-term viability of embryos are genetic or molecular and cannot be detected by simple microscopy. However, morphology is the best tool many embryologists have at hand. Informative and detailed assessments of embryo viability may require invasive impositions on the preimplantation embryo. That is why indirect non-invasive methods would be preferable. The literature review section of this study has reviewed the current morphological procedures routinely used in human assisted reproduction techniques, the biological basis that allow the selection of the best embryos, and the factors that influence our decisions. It also has taken a glance into the state-of-the-art approaches that can improve this selection as well as the different options available for in vitro fertilisation today.
The main study (Experiment 2) was aimed at determining whether the selection of bovine embryos for transfer based on morphology at around the 4-cell stage, a usual practice in human IVF clinics, is a good predictor of embryo viability. The hypothesis was that the selection (on Day 2 post-insemination) allows embryologists to choose the best embryos. To achieve this in a laboratory setting rather than by simulation, bovine embryos were used because of the difficulty of access to human embryos for regulatory and ethical reasons. Bovine eggs were studied, therefore, in a “human model” context.
A total of 220 embryos from 5 replicates were assessed on day 2 post-insemination according to the criteria used in a human IVF clinic, the Aberdeen Fertility Centre. Embryos were assigned to independent, randomly selected groups of ten, with each group of ten representing a different “patient”, 10 eggs being a reasonable yield to expect to get in a human clinic. Of these, 5 good or viable eggs were “selected”. In a real situation, from among eggs collected from a patient, normally two would have been transferred to the mother (in the Aberdeen Fertility Centre, as in most clinics in the UK, no more than 2 embryos are transferred to avoid multiple pregnancies) and three would have been cryopreserved for a future transfer (cryopreserved embryos have a lower survival and pregnancy rate because of the procedure). The other 5 embryos were termed “unselected” (and all or some or none may have been “non viable”). Stage of the embryos was also noted on days 4 and 6. Resultant blastocysts were classified blindly (i.e., operator unaware of whether they were “selected” or not) according to an adaptation of the criteria used by Lindner and Wright (1983) for bovine embryo morphology and evaluation. Day 7 blastocysts were assessed in terms of morphology (at x320 magnification), and 4 replicates out of 5 by a radioactive pyruvate uptake and utilisation test of metabolic activity of individual embryos. All of the blastocysts (5 replicates) were also fixed for cell counts with Hoechst stain (stains cells’ DNA) and observed under a fluorescence microscope (x400 magnification).
The medium used for culturing the embryos was SOFaaBSA until day 4 followed (as in sequential culture) by SOF+10%FCS+Vitamin E until day 7.
Before the main study (Experiment 2), preliminary studies (Experiment 1) were carried out with two batches of embryos to determine the time when most of the bovine embryos would be at the 4-cell stage, to ensure that timing of egg “selection” would be appropriate and to account for any differences between human and bovine embryo growth. During these preliminary investigations, and beginning at 36 hours post-fertilisation, embryos were assessed at intervals of 12 hours up to day 7 (post–insemination). Embryos were in three different treatments, as follows:
- SOFaaBSA followed by SOFaaBSA + 2% lipoprotein solution
- SOFaaBSA followed by SOF + 10% serum
Thus, differential growth and viability of embryos assigned to any of these three treatments could also be assessed. All of these blastocysts were also fixed for cell counts with Hoechst stain and observed under a fluorescence microscope (x400 magnification).
Preliminary data showed that the greatest prevalence of 3-4 cell embryos in SOFaaBSA occurred between 44 hours (43.1% of total embryos) and 56 hours post insemination (32.3% of total embryos), so the embryos were assessed during the remainder of the study (Experiment 2) on day 2 between 48 and 52 hours post-insemination. The presence of serum in Experiment I increased blastocyst yields (23.3% and 50% of total embryos under that treatment at 152 and 164 hrs post-insemination respectively). Use of lipoprotein supplement in SOFaaBSA supported blastocyst formation, especially between 152 and 164 hours (9.09% and 36.6% respectively). Unsupplemented SOFaaBSA had the lowest yields of blastocysts (6.15% and 21.54% at 152 and 164 hours). Thus sequential culture (SOFaaBSA followed by SOF+10% serum) proved to be the system that gave the best blastocyst yield.
Final results (Experiment 2) showed that Day 2 embryos that were selected gave higher yields (mean ± SEM) of blastocysts (1.14 ± 0.54 vs 0.32 ± 0.14 for the unselected ones) and also of grade 1 blastocysts on day 7 (0.5 ± 0.23 vs 0.14 ± 0.07). However, unselected embryos that reached blastocyst stage on day 7 showed no significant difference in terms of either cell counts (mean of selected group 72.93 ± 6.23 vs 69.71 ± 14.44 for unselected group) or diameter (171.58 μm ± 2.65 vs 170.42 μm ± 5.41) with the selected group. This showed that even though just a few developed, those unselected embryos that became blastocysts were comparable in quality and viability to blastocysts from selected embryos. These results are discussed in relation to the repercussions such findings, if they were to be echoed in human ART, could have ethically, scientifically and in relation to patients in a human IVF clinic, where embryos are selected for transfer and cryopreservation on day 2 and the unselected ones are discarded.