Introduction
In vitro fertilisation (IVF) is defined as a process used to assist with the conception of a child and involves a complex series of procedures. IVF is used to treat fertility and genetic problems (3). IVF has gained popularity over the past three decades with the birth of Louis Brown in 1978; the first test tube baby and is a relatively new technology (2). It is a process in which fertilisation is achieved in vitro which mimics in vivo environment.
In an IVF laboratory the gametes and embryos are exposed to various stresses throughout the different processes and stages of treatment at every level.
These occasions, the type of stresses and their effects are discussed in the following paragraphs.
1.Air quality
Air pollution and volatile organic compounds stress the gametes and embryos. It influences gametogenesis in both females and males, has negative impact on their quality on genetic as well as epigenetic level. Embryo development is affected resulting in abnormal fetal development and increased number of abortions. Effect of individual pollutant is not clearly known but it can be said that combination of oxidative stress, alterations in cell DNA and epigenetic alterations along with hormonal disturbances negatively impacts the fertility (7).
2.During Ovarian stimulation, Ovarian Hyper Stimulation Syndrome
Ovarian stimulation disrupts and delays the development of blastocysts (12, 39), influences perinatal outcomes due to its deleterious effects on embryo quality, oogenesis and endometrial receptivity (36) and delays the development of embryo (13). Exaggerated response to ovarian stimulation has detrimental effects on embryo morphology and quality (31, 32). Ovarian stimulation affects the number of mosaic/ aneuploidy embryos and impacts the quality of embryos (19).
3.During stimulation- in case patient develops fever
Hyperthermia negatively affects development of follicles and estradiol production by the ovaries (5).
4.Oocyte maturation- trigger to speed up the process
Oocyte maturation and meiosis is disturbed when the developing oocyte is exposed to supraphysiological levels of gonadotropins resulting in chromosomal disturbances (18). High doses of gonadotropins is indirectly proportional to the live birth rates (6). The type of GnRH analogue used influences extracytoplasmic defects significantly. Natural follicle development is impaired by GnRH agonist. Follicle development is variable in every case and so the dose of FSH administered varies from patient to patient (14).
5.Egg retrieval; aspiration pump
For oocyte retrieval ultrasound guided transvaginal aspiration is considered the gold standard procedure. Type of aspiration needle (wide or narrow bore or single or double channel), and pressure of the suction pump should be adequate. High aspiration pressure leads to denudation of cumulus cells around the oocytes and is commonly seen with use of large gauge needles (35).
6.Follicle flushing
Follicle flushing with high pressure is deleterious to the the follicle cells which support the endocrine luteal function (17, 21, 26, 34, 40). Better pregnancy outcomes and oocyte yield was observed in a study conducted by Kumaran A et al by direct oocyte retrieval at a pump pressure of 140 mmHg as compared to flushing and oocyte aspiration (25). Fluid flow rate, aspiration pressure and needle size are three important aspects of follicular flushing and they need to be carefully determined for a successful recovery without damaging the oocyte (35). High pressure removes the surrounding cumulus cells and damages the oocytes (28).
7.Surgical Sperm retrieval
Non obstructive causes- Testicular Sperm Aspiration (TESA), includes Testicular Fine Needle Aspiration (TFNA), Percutaneous Epididymal Sperm Aspiration (PESA)
Microsurgical Epididymal Sperm Aspiration (MESA), Percutaneous Biopsy (perc biopsy) of the testis.
Obstructive causes- Testicular Tissue Sperm Extraction (TESE), Microdissection TESE.
Cryopreservation (23)
8.Sperm freezing
Cryopreservation results in cryoinjury affecting sperm morphology, acrosome and the sperm tail, motility- sensitive to cryo damage, mitochondrial function and viability of the sperms (16, 41). Hence the proportion of viable sperms which are fully functional after cryopreservation is very low (20).
Sperm morphology
Significant reduction in sperms with normal morphology.
Rise in number of megalo, amorphous and elongated head.
Marked increase in midpiece abnormalities.
More incidence of loose heads and tails in cryopreserved sperms.
Sperm motility
Sensitive to cryo damage.
The motility parameters are decreased in freeze thawed sperms except ALH (10).
Cryopreservation results in alteration of the metabolic and functional status of the mitochondria and the cells as cell membranes are extensively damaged. High degrees of DNA fragmentation is observed in cryopreserved sperms (15).
9.Oocyte freezing
• DNA damage
• Aneuploidy
• Sister chromatid exchange
Poor results are seen as compared to embryo freezing accounting to low survival rates, fertilisation and development. Controversial and conflicting data is available regarding the effect of cryopreservation on oocytes due to limited number of studies
Microtubules of meiotic spindles in cryopreserved embryos are more prone to thermal changes immediately after thawing. These disturbances in the microtubular structures results in digyny, aneuploidies and arrest of cleavage after fertilisiation (8).
10.Embryo freezing
Results in mitochondrial DNA mutations. Plasma membrane characteristics, spindle system along with cortical granules may be responsible for the disparity in results of oocyte and embryo freezing with former being less practised. Chemical, mechanical and thermal forces account for cellular damage caused by cryopreservation of embryos and oocytes. Intracellular and Extracellular architecture may be compromised due to exposure to extreme conditions (11,37).
It is possible that the semen from fertile men has better resistance to cryopreservation due to the presence of antioxidants protecting against cryodamage.
Cryopreservation increases the susceptibility of DNA to denaturation by destabilizing the chromatin. In a study conducted by Lewis et al it was found that presence of antioxidants in semen of fertile men prevents damage by cryopreservation due to better resistance (27). However, contrasting data shows that DNA integrity is affected even in semen from fertile men after cryopreservation (10).
11.Laminar flow
Induces shear stress leading to polarisation of cytoskeleton of trophoblasts (cytoskeletal remodelling) by the movement of fluid. Chronic shear stress may trigger apoptosis and arresting of growth. Shear stress induces biphasic de novo synthesis of FOS protein and FOS proto- oncogene mRNA rapidly and MAPK8/9 (slower induction) which triggers TUNEL resulting in embryo death. SS is associated with apoptosis and lethality (42).
12.Changes in temperature, pH, osmolality in vitro are rapid and large as compared to those in the body which are relatively sudden and small. In a laboratory gametes and embryos also experience varying gas compositions. Constant temperature maintenance is required while handling gametes and embryos. Temperature fluctuations are very common and depends upon the fluid volume and the type of device being used- digital dry block heater, analog dry block heater, thermostat test tube heater (43). Therefore, perturbations can stress the embryos in an in vitro setting during the following occasions:
• Change of media, medium.
• Incubation- movement in and out of incubators.
• Movement of embryos and oocytes within the dish, and between dishes.
• Electronic witnessing system.
• During time lapse imaging
• Embryo checks.
• Embryo selection and transfer.
These fluctuations damage the meiotic spindle integrity and affects chromosomal organisation.
13.Metabolic stress-exposure to light
Gametes and embryos are exposed to light of varying intensities and sources in a clinical IVF laboratory during various procedures like oocyte retrieval, ICSI, morphology assessment, fertilisation checks, movement into dishes and incubators, embryo transfer (33). This affects the development and quality of embryo directly (toxicity) or indirectly (by photo oxidation). Exposure to light activates stress genes and damages the DNA by ionization.
TABLE 1 Sources of light in an ART laboratory and its impact
1. Windows
2. Ceiling Lights
3. Lamps
4. Microscopes
5. Direct effects of light
6. Indirect effects of light
7. Egg retrieval
8. Manipulation and routine handling
9. Embryo transfer
Filtered, curtained, shades, blinds
Fluorescent; cold white, warm white
Incandescent; dimmable and single intensity
Floor, desktop
Hoods/ cabinets
Inverted
Dissection
Time-lapse
Mineral oil overlay
Culture medium components
Plastic ware
ROS
Gene transcription
Culture medium breakdown products
Direct and indirect room lighting, hoods
Other surgery lamps and microscope
Direct and indirect room lighting, hoods
Other microscopes
Direct and indirect room lighting, hoods
Other microscopes
Headlamps, floor lamps, surgery lamps
Author
Object
Source
Parameter
Conclusion
Takahashhi et al 1999
Visible light 380-780nm, 400 lux
Hamster embryos
DNA fragmentation, evaluated by comet assay
Significant effect after ≥ 5 min illumination
Umaoka et al 1992
Visible fluorescent light, 2400 lux
Hamster embryos
Development of hamster 1 cell embryos to the 4 and 8 cell stage
Significant reduction after 5 min illumination. 30 min completely detrimental for the first cell cleavage
Schumacher and Fischer 1988
Visible fluorescent light 400-700 nm, 1600 lux
Rabbit embryos, day 1 post coitum
Cell proliferation by thymidine incorporation
Significant reduction after 30 min exposure
Barlow et al 1992
Halogen incandescent light, 4000 lux
Fertilised mouse oocytes
First cleavage, fertilised mouse oocytes
Significant reduction after 60 min illumination
Daniel 1964
Visible (UV and IR shielded) fluorescent light 2600 lux
Rabbit embryos
Number of cleavages pr time
Significant reduction after 4 hr illumination
Schumacher et al 1998
Visible fluorescent light <1600 lux
Rabbit day 3 and 4 embryos
DNA ploidy abnormalities
No increase in number after 24 hr illumination
Kruger and Stander 1985
Incandescent/ fluorescent visible light, 2900 lux
Mouse embryos
Development of 2 cell embryos to blastocysts
No effect after 30 min illumination
Squirrel et al 2001
Monochrome laser light 514, 532 and 568nm, 115kj/m2
Hamster embryos
Development of 2 cell embryos to morula/ blastocysts
Significant reduction at irradiance 115kj/m2
14.Sperm preparation- by repeated centrifugation, swim up, sperm wash, albumin columns and discontinuous percoll gradients. DNA integrity, motility and viability of the human sperm is determined by the quality of media used in sperm preparation (1). Density gradient centrifugation affects the DNA integrity, damaging the sperm DNA which is associated with low pregnancy outcomes (30).
15.Denudation
COCs are exposed to mechanical stress during denudation.
.
16.Embryo culture
Embryos are exposed to light, toxicants, volatile organic compounds, electromagnetic fields resulting in generation of reactive oxygen species.
17.PGD/PGS- biopsy during cleavage stage affects the viability of embryo and their implantation potential (9). Chances of vaginal infection and risk of acquiring Hyper Ovarian Stimulation Syndrome is more as more number of embryos are needed for testing. Embryos may be damaged during the process (4).
Procedures and techniques
18. ICSI
19. Assisted zona hatching (AZH), Laser assisted- improved implantation rates in cases of zona thickening in freeze-thawed embryos. But the procedure itself can be stressful for the oocytes. AZH also associated with premature hatching of the blastomeres.
20. Biopsy
21. Electronic witnessing system
22. Time lapse
23.Physical manipulation, rough handling and faulty technique
24. Exposure to plastic ware
25.Oxidative stress
External sources- Media, visible light, oxygen concentration, Freeze-thawing, ART procedures.
Internal sources- Gametes (oocytes and spermatozoa), embryos.
All these factors influence the IVF outcomes. There is increase in number of first trimester abortions because of altered fetal-placental ratio and abnormal fetal growth.
Prevention at the Laboratory level:
• Improving the air quality using highly efficient air filtration systems that filters air particles like microbes, perfumes as well as VOC can improve the IVF outcomes (22).
• Controlled ovarian stimulation prevents premature LH surge with the use of Progestin-primed ovarian stimulation (PPOS) (44).
• Maintaining an adequate pump pressure during oocyte retrieval (25).
• Temperature calibration- Follicle fluid is taken out in test tube warmer during aspiration, Test tubes are then brought to a pre heated station. Calibration certificates and contracts for machinery used in IVF laboratory.
• Use of antioxidants in the cryopreservation media.
• Preparation of sperms prior to freezing decreases apoptosis and improves the motility of sperms (38).
• Filtration can reduce the exposure to light by using filters (green bypass- Schott VG9) on microscopes, light fixtures for the removal of blue spectrum (deleterious to the embryos) and addition of antioxidants to the media for reducing oxidative stress caused by ROS. Use of Uv filters which are carefully calibrated and accurately controlled CO2 incubators.
• Care during handling can reduce stress caused by manipulation and rough handling.
• Reducing the oxidative stress by using antioxidants.
• Alarm systems in the laboratory to keep a check on the working of various equipments.
• Using Biological safety cabinets and ultrapure water systems.
Conclusion
Throughout the entire process of IVF, the gametes and embryos experience variable amounts and types of stresses which directly and indirectly impacts the outcomes of artificial conception. The exact pathophysiology of the effects seen due to exposure to stresses placed in a laboratory is still unclear due to lack of studies and data. More studies focussing around improving the human IVF outcomes need to be done