Add-ons in the ART Lab: Part 2

The start of life begins at the encounter of the sperm and the egg. They fuse and develop into a new creature with the help of oocyte activation.

The mechanism of egg activation is Ca2+-dependent. Ca2+ oscillation-inducing protein, phospholipase C zeta (PLCz), is introduced into the egg cytoplasm upon sperm-egg fusion. PLCz produces Inositol triphosphate (IP3). Then, Ca2+ is released from the endplasmic reticulum (ER) via IP3 receptor (IP3R). This Ca2+ rise triggers resumption of meiotic cell division, leading to the extrusion of second polar body and the formation of male and female pronuclei. Sperm contains PLCz in its sperm head. This is why the ICSI can induce Ca2+ release to resume the egg cell cycle. In ICSI failure case, the Ca2+ rise and oscillations were not been observed. In this context, two reason can be considered. One is deficiency of PLCz in sperm. Another is oocyte quality, expression level of IP3R and sensitivity of IP3 R for IP3 or Ca2+.

The result of cytogenetic analysis in ICSI oocytes without male or female pronucleus. Surprisingly 93% of oocytes were unfertilized that is oocytes were not activated.

Saunders, et al, reported that PLCz is a stron candidate of sperm factor, showing several evidences
1. PLCz triggers Ca2+ oscillations indistinguishable from those at fertilization.
2. PLCz-induced embryos can develop into blastocysts parthenogenetically.
3. PLCz removal from sperm extracts abolishes Ca2+ release in eggs.
4. PLCz content of a single sperm was sufficient to produce Ca2+ oscillations as well as a normal embryo.

From these experiments we answer the question “What is the best oocyte activation method?” The answer is optimal controlled ovarian stimulation (COS) to produce high-quality oocytes have abundant oocyte activations, that are, PLCZ and IP3.

In conclusion, currently PLCz is considered to be the most effective for oocyte activation. However, it requires high quality oocytes to be an effective activator.

ICSI was introduced into ART practice in 1992 to circumvent male factor infertility. Its use has now expanded to include other indications such as PGT, prior failed fertilisation via standard insemination and low oocyte number. Although ICSI is a critical technique employed in the majority of embryology laboratories it does involve the mechanical penetration of the oocyte with a bevelled/spiked pipette where the cytoplasm is aspirated into the micropipette and then injected back along with the sperm. This invasive procedure does make the oocyte susceptible to degeneration and also raises questions regarding the impact of aspiration on the cytoplasmic structure of the oocyte.

PIEZO-ICSI (a modified form of microinjection) has been used successfully in animal models for over 3 decades. PIEZO-ICSI uses a piezoelectric actuator to enable the micro-drilling of a blunt ended pipette into the oocyte and a piezo pulse is used to break the cytoplasmic membrane enabling deposit of a sperm without cytoplasmic aspiration. There are a limited number of studies demonstrating that PIEZO-ICSI increases fertilisation and decreases oocyte degeneration compared with standard ICSI. Despite PIEZO-ICSI being developed over 30 years ago (with clinical use in Japan for two decades), its widespread application has potentially been slowed by technical and laboratory safety concerns. This is because PIEZO-ICSI is technically more challenging compared with standard ICSI and requires the use of an operation fluid with inertial mass inside the microinjection pipette. This operation fluid, which is required to precisely control and transmit the PIEZO pulse, was initially mercury followed by fluorinert, neither of which are confirmed to be safe in a clinical context. There is now however a novel biocompatible operation liquid available and this lecture will cover the potential benefits of PIEZO-ICSI along with the results of using this biocompatible operation fluid in a recent clinical trial.

Inability of the embryo to implant is accounted for one of the main reasons for the failure of an IVF/ICSI cycle. A new technology to counter this obstacle emerged three decades ago; Assisted Hatching (AH). This aimed to facilitate the embryo in escaping from its outer coat i.e the zona pellucida and embed itself into the uterus. However, considerable debate surrounds the use of AH given the uncertainty that it results in significantly higher success rates. Its clinical justification is questioned. Hence, its routine application is not observed in most ART laboratories. The last Cochrane Review was published in 2013 with the conclusion that live birth rates (LBRs) are not significantly improved by AH. Here, we will explore the scientific data that has been presented since then. The potential indications for the application of AH will be discussed; previous repeated implantation failure, poor prognosis patients, and advanced maternal age (AMA). While new points regarding the topic have been highlighted, the question of the clinical justification of AH remains largely unanswered. The potential harmful effects in terms of epigenetics should be seriously considered although those are also yet to be elucidated. Hence, assisted hatching stands amongst the rest of the add-on treatments in IVF which are also controversial given the uncertain evidence of their significant impact on the live birth rates.