Looking for the best in show

22 January 2020:

For a couple unable to naturally conceive, in vitro fertilisation is often the only option – but it’s one that involves months, even years, of hope intertwined with disappointment. Contributing to the ‘hit and miss’ are the eggs taken from fallopian tubes of hopeful mothers or donors: healthy eggs are essential for healthy embryos to develop into a pregnancy and, ultimately, a baby.

“I have two aunts who were unable to have children,” said Hanna McLennan, a PhD student in obstetrics and gynaecology at CNBP’s University of Adelaide node. “It’s just such an important thing in people’s lives that can define their whole existence.”

Working in the centre’s Reproductive Success group under Prof Jeremy Thompson, McLennan is working to develop a non-invasive way to identify healthy eggs, or oocytes, at the point of extraction, using chemical probes and optic fibres developed at CNBP.

Currently, when immature oocytes are harvested for IVF, they are grown in a medium with sperm to turn them into embryos. “But, if we’ve got a group of 10 embryos, we’re very limited in how much we can know about which embryo is the best one,” said McLennan.

“At the moment, all we can do is look at an embryo [under the microscope] and see if it has divided the right amount of times for how long it’s been developing, and if it looks aesthetically ‘good’.

“But at the same time, there’s complex metabolism going on in these cells, and they’re producing signals that they release into the media they’re in. Is there a way we can detect these signals immediately? Or develop a probe we can put next to the cells and measure a signal?”

Turns out, you can. In November 2019, McLennan was the co-lead author in a scientific paper in the Journal of Biophotonics that successfully demonstrated how the pH (acidity or alkalinity) of an oocyte could be measured in just a drop of surrounding medium, using a new optical fibre probe they’ve dubbed OPF1. Importantly, this was done without affecting or disturbing the egg.

Co-lead author Avishkar Saini positions the OFP1 probe next to the egg. Credit: Georgina Sylvia.

“The pH is a reflection of cell metabolism,” explained McLennan. “When cells are taking glucose and turning it into energy, they produce as a byproduct lactic acid, which slightly decreases pH, and if we can detect pH, we can measure metabolism. Also, pH changes would potentially be higher [or more acidic] in stressed cells, so it may suggest that egg is less healthy. And then, more generally, the eggs need a very specific pH level to grow.”

The OPF1 fibre is just 0.2 mm in diameter, only slightly bigger than the developing cumulus-oocyte complex it is trying to measure. OPF1 is connected to an LED that flashes two pulses at different wavelengths, which excite a chemical fluorescent tip that measures the wavelength of light bouncing back, giving a precise count of how many hydrogen ions are in the medium, thereby determining its pH.

The team not only successfully measured pH in an oocyte, but proved OPF1 was able to detect changes in pH in response to stress (the adding of cobalt chloride), and changes in pH due to accelerated development (the adding of follicle stimulating hormone, or FSH).

“We’ve proved the probe works, but there are also lots of different signals from different stages of development [of embryos] we’d like to measure,” said McLennan. “This study tells us that the signal we get is consistent and strong enough to detect a biological level of change. And while there’s a lot of work in chemistry and physics to develop other probes, we now know that it’s worthwhile, and now it’s basically working out what the next targets are.”

Journal: Journal of BIOphotonics

Publication Title: A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus‐oocyte‐complex (COC)

Authors: Hanna J. McLennan, Avishkar Saini, Georgina M. Sylvia, Erik P. Schartner, Kylie R. Dunning, Malcolm S. Purdey, Tanya M. Monro, Andrew D. Abell, Jeremy G. Thompson

Summary: Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non‐invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell‐safe with no inherent cell toxicity or light‐induced phototoxicity indicated by negative DNA damage staining. Pre‐measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01‐0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

Link: https://onlinelibrary.wiley.com/doi/abs/10.1002/jbio.201960038