Abstract
The IVF laboratory is central to Medically Assisted Reproduction perhaps representing also the most crucial extrinsic factor in determining success or failure of treatment. Current science and technology are unable to improve the intrinsic developmental potential of gametes. Therefore, the “mission” of the IVF laboratory consists in the ability to preserve the innate characteristics of sperm and oocytes in the course of preimplantation development and minimize the possible detrimental impact of diverse forms of manipulation. To this end, during culture and manipulation, physical factors (e.g., temperature, atmosphere composition) and stressors (e.g., oocyte microinjection, embryo biopsy) should be monitored and controlled, in order to guarantee stability of conditions considered to be the most appropriate to support and facilitate gamete and embryo function in vitro. In this scenario, the human factor and effectiveness of technical equipment contribute in similar or equivalent proportions in determining clinical outcome. In light of this, not only is monitoring of working conditions of equipment important, but objective assessment of key segments of the IVF process is vital. Performance indicators respond to this need, offering specific, important, and objective measurements of essential processes, such as fertilization, development to blastocyst stage, and cryopreservation. Thus, critical analysis and interpretation of indicators can lead to consistency of results and continued improvement.
10.1 Introduction
The IVF laboratory is central to Medically Assisted Reproduction perhaps representing also the most crucial extrinsic factor in determining success or failure of treatment. Current science and technology are unable to improve the intrinsic developmental potential of gametes. Therefore, the “mission” of the IVF laboratory consists in the ability to preserve the innate characteristics of sperm and oocytes in the course of preimplantation development and minimize the possible detrimental impact of diverse forms of manipulation. To this end, during culture and manipulation, physical factors (e.g., temperature, atmosphere composition) and stressors (e.g., oocyte microinjection, embryo biopsy) should be monitored and controlled, in order to guarantee stability of conditions considered to be the most appropriate to support and facilitate gamete and embryo function in vitro. In this scenario, the human factor and effectiveness of technical equipment contribute in similar or equivalent proportions in determining clinical outcome. In light of this, not only is monitoring of working conditions of equipment important, but objective assessment of key segments of the IVF process is vital. Performance indicators respond to this need, offering specific, important, and objective measurements of essential processes, such as fertilization, development to blastocyst stage, and cryopreservation. Thus, critical analysis and interpretation of indicators can lead to consistency of results and continued improvement.
10.2 Monitoring
10.2.1 Culture Conditions
In vivo, the microenvironment provides ideal homeostatic conditions for preimplantation development. However, the scenario is completely different when the in vitro-derived embryo develops in a culture droplet placed in a Petri dish. Several physical parameters need to be controlled for carefully to obtain a healthy embryo in vitro. Providing and maintaining proper culture conditions is crucial to minimize stress imposed upon gametes and embryos and to optimize the in-vitro environment.
The major relevant physical parameters are, but not limited to, temperature, pH, osmolarity, humidity, and air quality. (See, e.g., Chapters 2 and 6 for details.)
10.2.1.1 Oxygen
In 1979 an association between mammalian embryo development in vitro and oxygen levels was first reported (Morriss and New, 1979). High and nonphysiological oxygen concentrations may have a negative impact through oxidative stress, causing a defective embryo development, with higher rates of fragmentation (Bedaiwy et al., 2004). Conversely, too low oxygen levels may impair embryogenesis, with an involvement of several oxidative processes (Ng et al., 2018). For many years, in human IVF laboratories, atmospheric oxygen tension (~20%) has been used in embryo culture. Therefore, for these reasons, modern IVF incubators should allow monitoring and regulation of oxygen concentration (Swain et al., 2014).
10.2.1.2 Temperature
Temperature is one of the most important factors impacting on IVF outcome. It is well known that temperature can affect gamete and embryo function, particularly meiotic spindle stability (Sun et al., 2004; Wang et al., 2001; Wang et al., 2002) and, under specific conditions, embryo metabolism (Leese et al., 2008).
Laboratory equipment designed to maintain a desired temperature during culture should be subject to daily (if not continuous) monitoring. Indeed, annotation of incubator temperature before start of daily laboratory procedures should be a standard practice. It is common notion that to achieve undisturbed development and desired clinical outcomes, embryos require stable physical conditions when cultured in an incubator (Boone, 2010; Swain, 2014).
During embryo assessment outside the incubator, embryos are exposed to room temperature (Wang, 2002), although great care is normally taken to minimize temperature fluctuations by reducing the time required for observation using warm microscope stages (Magli, 2008).
10.2.1.3 pH and Osmolality
pH is an important regulator of metabolism and other cell functions. Therefore, its dysregulation can severely affect gamete/embryo function and impact IVF outcome. Unlike embryos that have the capacity, although limited, to regulate their internal pH (pHi), oocytes have no robust regulatory mechanisms. Therefore, careful control and monitoring of external pH (pHe) at isolated time points, i.e., testing samples of culture medium exposed to the same conditions, is imperative in IVF (Swain, 2010).
For culture in vitro, pH buffers have been included in the formulation of media to help stabilize pHe (Will et al., 2011) and thereby minimize deleterious intracellular changes arising from fluctuations in pHi (Phillips et al., 2000).
The capacity of embryos to regulate pHi emerges from various studies showing that embryos can be cultured over a pHe range of pH 7.0–7.4 in the absence of significant effects on development (John & Kiessling, 1988; Lane et al., 1998), while deviations of pHe outside this range have shown detrimental effects (Leclerc et al., 1994; Zhao & Baltz, 1996; Zhao et al., 1995; Lane & Bavister, 1999; Lane et al., 1999a,b). At the moment, the optimal pHe value to achieve in the culture milieu is not known, as also shown by the wide range of pHe reported in information sheets of different commercial media.
Another key parameter is osmolality, which should range from 280 to 285 mOsm/kg of water. This information is provided by the manufacturers and considered reliable. However, independent measurement may be required when different solutions are mixed, e.g., when volumes of concentrated protein solutions are diluted in protein-free culture media. Osmolality over 300 mOsm/kg can inhibit embryo development, although this effect may be mitigated by amino acids that act as osmolytes (Swain, 2019).
10.2.1.4 Humidity
Humidity of the incubator environment can be a significant factor in determining IVF outcome. Both for oocyte and embryo culture, 90–100% humidity is considered optimal in an incubator environment.
Appropriate humidity levels minimize the risk of water evaporation from culture drops. If medium evaporation occurs, pH and solute concentrations increase and embryos are no longer exposed to the same solute concentrations initially present in the culture microenvironment. This possible change in osmolality can have detrimental consequences for embryo development (Lane et al., 2008; Swain et al., 2012).
Therefore, the more appropriate and specific conditions to prevent water evaporation from culture drops should be identified and adopted for every laboratory.
10.2.1.5 Air Quality
An additional environmental factor that impacts embryo culture conditions is air quality. Presence and abundance of volatile organic compounds (VOCs) in the laboratory and/or in the incubator may compromise embryo development in terms of cleavage rate and implantation potential (Cohen et al., 1997; Hall et al., 1998; Merton et al., 2007; Khoudja et al., 2013).
VOCs have been detected in gas supply tanks used for IVF incubators (Hall et al., 1998). In such cases, filtering the gases through inline filters before entering the incubator may be an effective approach to improve incubator atmosphere. These filters not only contain HEPA filtration systems to reduce particle counts, but they can also reduce VOC through activated charcoal- or potassium permanganate-based filters.
In this regard, periodic checks of particle count, VOCs, filter integrity, and microbial count should be carried out, in areas where gametes and embryos are handled.
10.3 Performance Indicators
The role of the IVF laboratory in the overall process of MAR is fundamental, involving procurement, preparation, and measurement techniques, aiming at generating a biological product (the embryo) from processing of cells types (the male and female gametes). This procedure in vitro is highly sensitive to extrinsic conditions and also profoundly affected by patient characteristics. For these reasons it has been difficult to standardize and to measure performance in a structured fashion. There have been some efforts by national societies to set standards for benchmarking. For example, the Association of Clinical Embryologists defined several performance indicators for the IVF laboratory, although in a document more generally dedicated to IVF good practice and guidelines (Hughes et al., 2012). In 2012, performance indicators for gamete and embryo cryopreservation were described in a consensus paper published by Alpha Scientists in Reproductive Medicine (2012). In 2017 a consensus document, from ESHRE and Alpha Scientists in Reproductive Medicine, reporting performance indicators (PI) applicable to the mainstream process of the IVF laboratory was published (ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine, 2017). In 2018, ASEBIR (the Spanish embryology association) critically assessed the above cited ESHRE–Alpha document (Lopez-Regalado et al., 2018), stating that some competence levels were not realistic and not based on state-of-the-art performance. In addition, they suggested inclusion of more variables, e.g., embryo utilization rate.
10.3.1 Definition and Characteristics of Performance Indicators
Within the general framework of a Quality Management System (QMS), PIs are measurable parameters to assess the quality of essential healthcare objectives; e.g., patient safety, treatment efficacy, and efficiency. In the specific context of a clinical laboratory, PIs are required to appraise the laboratory’s specific contribution to patient treatment.
In order to provide significant information and be generated routinely, any PI should respond to several criteria:
Coverage of the most important phases of the process occurring in the IVF laboratory
Definition of specific biological or technical step to be monitored
Identification of qualifiers (e.g., female age), confounders (e.g., day of embryo transfer), and endpoints (e.g., development to blastocyst stage)
Reliability and ease of measurement
Ease of data collection
Numerical expression by an unambiguous and predetermined formula.
Among PIs, key performance indicators (KPIs) are especially important to:
Assist in the introduction of a new technique or process
Define minimum proficiency standards
Monitor laboratory performance over time
Define internal quality control and external quality assurance programs
Set benchmark and quality improvement objectives.
10.3.2 General Recommendations for Indicators of Fresh MAR Treatments
10.3.2.1 Frequency
In clinics with steady and high workload, indicators should be collected monthly. However, in clinics with low monthly activity, such a time interval is not applicable because PI values would not be reliable. Therefore, longer periods between consecutive measurements or a minimum number of treatments (at least 30) should be considered, depending on the stability of the indicator in question.
10.3.2.2 Types of Indicator
Based on their importance and relevance, indicators of the IVF laboratory may be classified into:
i. Reference indicators: relevant to patient response to ovarian stimulation, thereby potentially able to provide indirect information on oocyte quality.
o Proportion of oocytes recovered (stimulated cycles)
o Proportion of MII oocytes at ICSI.
ii. PIs: relatively less important and therefore not necessary to be reported in control charts, but should be documented and stored.
o Sperm motility post-preparation (for IVF and intrauterine insemination)
o IVF polyspermy rate
o 1 PN rate (IVF)
o 1 PN rate (ICSI)
o Good blastocyst development rate.
iii. KPIs: crucial to monitor the most important steps of the IVF process and therefore demanding careful analysis.
o ICSI damage rate
o ICSI normal fertilization rate (2PN and 2PB)
o IVF normal fertilization rate (2PN and 2PB)
o Failed fertilization rate (IVF)
o Cleavage rate (day 2)
o Day 2 Embryo development rate (4-cells)
o Day 3 Embryo development rate (8-cells)
o Blastocyst development rate (day 5)
o Successful biopsy rate
o Blastocyst cryosurvival rate
o Implantation rate (cleavage-stage)
o Implantation rate (blastocyst-stage).
Notably, different KPIs have different significance and reliability. For example; ICSI damage/fertilization rates are very reliable and informative of operator performance; IVF fertilization rates are more relevant to gamete quality and handling; cleavage and blastocyst development rates are very informative of the performance of the culture system; implantation rates, although very important, are less reliable because more influenced by the typology of the reference population, as well as clinical protocols and procedures.
10.3.2.3 Reporting
Indicators should be reported and assessed in relation to proficiency and benchmark values that define a “desirable range” for the laboratory performance.
10.3.2.4 Influence of Clinical and Laboratory Procedures
Clinical (e.g., time of oocyte retrieval from trigger of final maturation, typically 36–38 hours) or laboratory (timing of embryo assessment) procedures can impact the value of indicators. Such factors should be taken into consideration.
10.3.2.5 Reference Population
Several indicators are certainly influenced by the characteristics of the patient population from which they are collected. The ESHRE/Alpha consensus document identified a “reference population” responding to the following criteria:
female patients <40 years old
own fresh oocytes
ejaculated spermatozoa (fresh or frozen)
no PGT
both insemination methods (i.e. standard IVF and ICSI).
Individual clinics may opt for a different (or multiple) reference population that may better represent the typology of their patient.
10.3.3 Indicators of Cryopreserved MAR Treatments
KPIs for cryopreserved MAR treatments were published by Alpha Scientists in Reproductive Medicine in 2012 (Alpha Scientists in Reproductive Medicine, 2012). Most of the general criteria of definition, characteristics, and recommendations reported above and relevant to fresh treatments are also applicable to cryopreservation PIs. Such indicators are divided into categories relevant to the use of cryopreserved oocytes, zygotes, cleavage-stage, and blastocyst-stage embryos.
i. Morphological survival
ii. Fertilization rate
iii. Embryo development rate
iv. Implantation rate
i. Morphological survival
ii. Cleavage rate
iii. Embryo development rate
iv. Implantation rate
i. Morphological survival: fully intact
ii. Morphological survival: ≥50% intact
iii. Post-warming embryo development rate (if applicable)
iv. Implantation rate
i. Morphological survival
ii. Embryo transfer rate
iii. Implantation rate
Like PIs of fresh treatments, cryopreservation PIs relevant to survival and subsequent development of oocytes and embryos should be reported and appraised in comparison with “desirable ranges” of laboratory performance defined by proficiency and benchmark values. Importantly, such proficiency and benchmark values are different depending or whether gametes/embryos are cryopreserved by slow freezing or vitrification. However, cryopreservation KPIs are based on evidence or empirical experience generated until 2010, i.e., a period when vitrification had been already introduced on quite a large scale, but not probably optimized. Since then, vitrification has experienced further expansion and standardization. In the light of such progress, some proficiency or benchmark values appear inappropriate in consideration of current standards of performance. For example, in the Alpha consensus document, the competence (proficiency) value of blastocyst survival rate after cryopreservation by vitrification is 80%, clearly inadequate to appraise the current standard of performance of blastocyst cryopreservation in the large majority of laboratories worldwide. Similarly, a survival rate of 70% as competence value for oocyte vitrification appears too low compared with data reported in publications of the last several years. More generally, assessment of oocyte survival rate may be particularly prone to error. For example, for a given cohort of oocytes this rate may vary significantly depending on whether morphological viability is assessed shortly after the warming procedure or 60–90 minutes later. Therefore, while the Alpha cryopreservation KPIs document has been instrumental in measuring laboratory performance and, more generally, in introducing a methodology for objective evaluation, significant progress in oocyte and embryo cryopreservation calls for a review of proficiency and benchmark values.
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