Chapter 13 – Micromanipulation Techniques




Abstract




Biologists and physiologists began to micromanipulate cells during the last century, using a variety of manipulator systems to dissect or record from cells. The earliest attempt to inject sperm was recorded in 1914, when G.I. Kite injected sperm cells into starfish oocytes, but with inconclusive results (Lillie, 1914). Experiments in which sperm were injected into eggs around the mid-1960s were primarily designed to investigate the early events of fertilization, i.e. the role of membrane fusion, activation of the oocyte and the formation of the pronuclei. Two series of early experiments by independent groups demonstrated major species differences. Hiramoto showed that microinjection of spermatozoa into unfertilized sea urchin oocytes did not induce activation of the oocyte or condensation of the sperm nucleus (Hiramoto, 1962), whereas others demonstrated the opposite in frog oocytes. Ryuzo Yanagimachi and his group later demonstrated that isolated hamster nuclei could develop into pronuclei after microinjection into homologous eggs, and a similar result was obtained after injecting freeze-dried human spermatozoa into a hamster egg (reviewed by Yanagimachi, 2005). These experiments indicated that membrane fusion events can be bypassed during activation of mammalian oocytes, without compromising the initiation of development. The experiments not only provided information on the mechanism of fertilization, but also led to a new technique in clinical embryology.





Chapter 13 Micromanipulation Techniques




Introduction


Biologists and physiologists began to micromanipulate cells during the last century, using a variety of manipulator systems to dissect or record from cells. The earliest attempt to inject sperm was recorded in 1914, when G.I. Kite injected sperm cells into starfish oocytes, but with inconclusive results (Lillie, 1914). Experiments in which sperm were injected into eggs around the mid-1960s were primarily designed to investigate the early events of fertilization, i.e. the role of membrane fusion, activation of the oocyte and the formation of the pronuclei. Two series of early experiments by independent groups demonstrated major species differences. Hiramoto showed that microinjection of spermatozoa into unfertilized sea urchin oocytes did not induce activation of the oocyte or condensation of the sperm nucleus (Hiramoto, 1962), whereas others demonstrated the opposite in frog oocytes. Ryuzo Yanagimachi and his group later demonstrated that isolated hamster nuclei could develop into pronuclei after microinjection into homologous eggs, and a similar result was obtained after injecting freeze-dried human spermatozoa into a hamster egg (reviewed by Yanagimachi, 2005). These experiments indicated that membrane fusion events can be bypassed during activation of mammalian oocytes, without compromising the initiation of development. The experiments not only provided information on the mechanism of fertilization, but also led to a new technique in clinical embryology.


During the late 1980s Jacques Cohen and colleagues developed a microsurgical technique to aid fertilization of human oocytes via partial dissection of the zona pellucida (PZD) (Cohen et al., 1988). This mechanical technique involves breaching the zona pellucida with a sharp glass micropipette to create a slit and subsequently placing the dissected oocyte into a suspension of spermatozoa, on the assumption that sperm entry is facilitated by the slit. Lanzendorf et al. (1988) demonstrated formation of pronuclei after direct injection of sperm into human oocytes, and in 1989 S.C. Ng and colleagues in Singapore reported the first pregnancy after inserting several spermatozoa into the perivitelline space, subzonal sperm injection (SUZI). They later also reported activation of human oocytes following intracytoplasmic injection (ICSI) of human spermatozoa (Ng et al., 1991).


In 1992, Palermo and colleagues in Brussels reported the first live birth from this technique of ICSI (Palermo, 1992). Since the time of these pioneering reports, ICSI is now a standard and successful assisted reproduction treatment, with >2.5 million babies born worldwide by 2012.


The technique of assisted hatching was also developed during the 1990s, using micromanipulation to cut a slit in the zona pellucida or dissolve a hole in the zona with an acid solution (zona drilling, ZD). Assisted hatching was proposed as a means of facilitating embryo implantation in selected cases. Figure 13.1 shows a diagrammatic representation of these micromanipulation procedures.





Figure 13.1 Micromanipulation techniques include subzonal injection (SZI), intracytoplasmic sperm injection (ICSI), zona drilling (ZD) and partial zona dissection (PZD).



ICSI – Intracytoplasmic Sperm Injection


Prior to 1992, the majority of cases of severe male infertility were virtually untreatable, and failure of fertilization was observed in up to 30% of IVF treatments for male infertility. The introduction of micromanipulation techniques such as PZD, SUZI and ZD raised the hopes of a better prognosis for these cases, but did not overall provide a substantial improvement in success rates. The introduction and successful application of ICSI by Gianpiero Palermo and colleagues at The Free University in Brussels, Belgium, produced a dramatic improvement in the treatment of severe male infertility by ART.



Genetic Implications


The establishment of ICSI as a routine technique was quickly followed by the introduction of techniques for collecting sperm samples from the epididymis and directly from the testis, so that the whole spectrum of male infertility can now be treated, from suboptimal ejaculate samples or ejaculatory failure, to obstructive and nonobstructive causes of azoospermia. However, an increasing number of genetic defects have been found to be associated with male infertility: a higher incidence of numerical and structural chromosomal aberrations is found in infertile and subfertile men than in the general population, in particular karyotypes 47XXY, 47XYY, 46XX, 46X, derY, Robertsonian translocations, reciprocal translocations, inversions and additional marker chromosomes. Between 12% and 18% of men with azoospermia or severe oligospermia (less than 300 000 sperm in the ejaculate) have deletions in intervals 5 and 6 on the long arm of the Y chromosome. Microdeletions of the q11 region of the Y chromosome are related to the dysfunction of Deleted in Azoospermia (DAZ) and RNA-binding motif (RBM) genes, and androgen receptor (AR) gene mutations have also been reported in infertile men. In a population of approximately 3000 infertile men, the pathological (nonpolymorphic, phenotype associated) microdeletions rate, in at least one of four critical regions on the Y chromosome, was found to be as high as 22%, with an additional (as yet unknown) percentage being attributed to cryptic mosaicism. Furthermore, it appears that microdeletions will be transmitted in at least 10% of unselected father/son pairs.


The Belgian group responsible for the first ICSI births have carried out prospective follow-up studies of their first cohort of ICSI babies, born between 1992 and 1996; they confirm that this group of boys has normal pubertal development and adequate Sertoli and Leydig cell function. They then carried out a study of semen parameters in a small cohort of young adults aged between 18 and 22 years who were conceived after ICSI for male infertility with fresh ejaculated sperm; this group of young men had significantly lower median sperm concentration, total sperm count and total motile sperm count when compared with a control peer group born after spontaneous conception. Although no clear correlation was seen between the semen parameters of the young men in the study and their fathers, the study confirms the need for further investigation of transgenerational passage of male infertility (Belva et al., 2016).


Three to ten per cent of infertile men present with congenital bilateral absence of the vas deferens (CBAVD), and approximately 65% of these individuals carry the gene for cystic fibrosis (CF), with defects in the cystic fibrosis conductance regulator (CFTR) gene. Many are compound heterozygous for the CFTR mutation, with an increased risk of having children with CF or CBVAD.


Although the genetic risk for couples who require ICSI treatment has yet to be fully defined, karyotyping, and preferably also Y-microdeletion analysis, is recommended as part of the pretreatment screening process for men with severe male factor infertility referred for ICSI (Qureshi et al., 1996; Simoni et al., 1998). The couple should also have access to professional genetic counseling to discuss potential risks, and appropriate informed consent must be obtained before treatment.



Surgical Sperm Retrieval


In cases of obstructive azoospermia, samples can be aspirated from the epididymis. The original ‘open’ microsurgical technique of microepididymal sperm aspiration (MESA) was superseded by the simpler procedure of percutaneous epididymal sperm aspiration (PESA), which can be carried out by fertility specialists without microsurgical skills, and can be performed under local anesthetic or mild sedation as an outpatient procedure. Aspiration is carried out using a 25-gauge butterfly needle connected to a syringe. If sperm cannot be aspirated from the epididymis, a modification of the technique using wide-bore needle aspiration of the testis, testicular sperm aspiration (TESA) or testicular fine needle aspiration (TEFNA) often harvests sufficient testicular spermatozoa to carry out an ICSI procedure. In nonobstructive azoospermia, spermatogenesis is impaired. The epididymis is devoid of sperm, but the testis usually contains focal areas of spermatogenesis: this focal spermatogenesis makes diagnosis based upon a single biopsy unrealistic, and multiple biopsies may be required. Prepared testicular samples can also be cryopreserved for a future ICSI procedure at the time of diagnostic testicular biopsy. The biopsy is carried out either by multiple needle aspirations (TEFNA or TESA) or by open biopsy (testicular sperm extraction: TESE), and both procedures may be safely carried out with local anesthetic or mild sedation.



Indications for ICSI


The ICSI procedure involves injecting a single immobilized spermatozoon directly into an oocyte, and therefore it can be used not only for cases in which there are extremely low numbers of sperm, but in bypassing gamete interaction at the level of the zona pellucida and the vitelline membrane it can also be used in the treatment of qualitative or functional sperm disorders.




  1. 1. Couples who have suffered recurrent failure of fertilization after IVF-ET may have one or more disorders of gamete dysfunction in which there is a barrier to fertilization at the level of the acrosome reaction, zona pellucida binding or interaction, zona penetration, or fusion with the oolemma. ICSI should be offered to patients who have unexplained failure of fertilization in a previous IVF–ET cycle.



  2. 2. Severe oligospermia can be treated with ICSI; in patients where as many normal vital sperm can be recovered as there are oocytes to be inseminated, fertilization can be achieved in approximately 90% of cases. In extreme cases of cryptozoospermia, where no sperm cells can be seen by standard microscopy, centrifugation of the neat sample at higher than usual centrifugal force (1800 g, 5 minutes) may result in the recovery of an adequate number of sperm cells.



  3. 3. Severe asthenozoospermia, including patients with sperm ultrastructural abnormalities such as Kartagener’s syndrome, or ‘9 + 0’ axoneme disorders can be treated by ICSI.



  4. 4. Teratozoospermia, including absolute teratozoospermia or globozoospermia.



  5. 5. In cases of CBAVD, vasectomy or postinflammatory obstruction of the vas, sperm samples can be retrieved by PESA, TESA or TESE.



  6. 6. Samples can be recovered by needle or open biopsy of the testis in cases of nonobstructive azoospermia.



  7. 7. In cases of ejaculatory dysfunction, such as retrograde ejaculation, a sufficient number of sperm cells can usually be recovered from the urine.



  8. 8. Paraplegic males have been given the chance of biological fatherhood using electro ejaculation and IVF; they may also be successfully treated using a combination of PESA/TESA/TESE and ICSI.



  9. 9. Immunological factors – couples in whom there may be antisperm antibodies in female sera/follicular fluid or antisperm antibodies in seminal plasma following vasectomy reversal or genital tract infection can be successfully treated by ICSI.



  10. 10. Oncology – male patients starting chemotherapy or radiotherapy should have semen samples frozen for use in the future; ICSI offers the patient an excellent chance of achieving fertilization following recovery from their disease and treatment. Testicular biopsy specimens may also be cryopreserved for these patients as a further back-up when the quality of the ejaculate is inadequate for freezing.



  11. 11. For preimplantation genetic diagnosis involving DNA amplification by PCR, ICSI should be used as the means of fertilization to prevent sperm contamination of the sample.


Although the main indication for ICSI was originally for the treatment of male factor infertility, its use has become far more widespread, with an increasing trend for its routine use in treating indications that include moderate male subfertility, advanced maternal age, low responder patients, and donor oocytes or sperm; indeed, some clinics now use ICSI as a routine for all indications. European data for 2005 showed that the proportion of ICSI cycles in different countries ranged from 58% to 67%; in the USA, 62.2% of fresh nondonor cycles used ICSI during 2006. A review of European data collected between 1997 and 2011 confirmed that the proportion of ICSI versus IVF cycles continued to increase, reaching a plateau of around 75% in 2008 (Ferraretti et al., 2017), and in 2016 the International Committee for Monitoring Assisted Reproductive Technologies reported that global use of ICSI remained constant at around 66% of nondonor cycles (Dyer et al., 2016). Several randomized controlled studies have compared the efficacy of IVF versus ICSI in couples with non-male factor infertility, with results that showed no difference in fertilization or pregnancy rates. A Cochrane review (van Rumste et al., 2004) concluded that the use of ICSI for non-male factor infertility remains an open question, and further research should focus on live birth rates and adverse events. In their 2008 report on good clinical treatment in assisted reproduction, the European Society for Human Reproduction and Embryology (ESHRE) published an Executive Summary that concludes: ‘ICSI should be considered in the presence of severe sperm abnormalities or a history of fertilization failure in conventional IVF attempts. It must be emphasized that ICSI does not represent the most suitable treatment for female pathologies such as poor ovarian response or previous implantation failure.’ This conclusion continues to be reinforced by data reported up to the present day.



Practical Aspects


ICSI demands the same meticulous attention to detail that is needed in all IVF manipulations, but the number of details requiring attention is dramatically increased. Successful results with ICSI can only be achieved with the dedication of concentrated time, effort and patience.



Location of ICSI Set Up

The laboratory should preferably be on a ground floor, near a structural frame or wall to minimize vibrations, and must be kept dust-free. The equipment must be installed on a substantial bench top, away from distractions of traffic such as people or trolleys, etc. Any vibration will interfere with the injection procedure, and it is essential to make sure that the equipment is completely stable, using anti-vibration equipment if necessary. Subdued lighting is helpful for microscopy. Well in advance of any ICSI procedure, ensure that the microscope’s optics are checked. Ensure that the tool holders and all other parts of the micromanipulation system are correctly fitted and adjusted for optimal range of movement, and that the microtools can be accurately aligned.



Microinjection Equipment

All the major microscopy companies now supply microinjection set-ups ready for use. The essential element is an inverted microscope with ×10, ×20 and×40 objectives, with Hoffman modulation contrast optics in order to visualize the cells on plastic Petri dishes (Nomarski optics uses polarized light and cannot be used through plastic). The micromanipulators consist of two coarse motorized manipulators and two fine mechanical, electrical or hydraulic joysticks, together with microsyringes capable of delivering minute quantities of liquid. Tables 13.1 and 13.2 compare the different types of equipment currently available. For training purposes, it is advisable to have a camera attached to one of the microscope optical outlets.




Table 13.1 Micromanipulator







































Manipulator type Pros Cons Examples
Manual
Fluid-filled: oil, fluorinert, distilled water Easy control Air bubbles render control difficult. Leakage of fluid can be messy, requires careful priming. Time-consuming to set up and flush system. Glass syringes expensive Narashige, Eppendorf
Air-filled OK for holding pipette Good control only if set up carefully, with a bubble of oil between air and media Research Instruments, Narashige
Pneumatic Injection controlled by foot pedal Pressure leakage Tritech
Piezo Drill Less disruption to the cytoplasm Difficult learning curve; possible use of mercury PrimeTech


Comments contributed by M. Blayney, A. Burnley, L. Devlin, D. Kastelic, M. van den Berg and B. Woodward.



Table 13.2 Joysticks







































Joystick type Pros Cons Examples
Hanging joystick Comfortable to use for short cases Elevated hand position can be uncomfortable during long ICSI cases Narashige
Standing joystick All-in-one 3D control May fall aside, with possible oocyte damage Narishige, Eppendorf
Mechanical Separate coarse and fine manipulators. Very little to go wrong Usually fixed to the microscope, with possible vibration transition; 2D control separate to ‘up-down’ control Research Instruments
Hydraulic Smooth System fails if there are any leaks; the oil degrades in sunlight Narashige
Motorized Very convenient if positions can be stored in memory. Movements are quick, decreasing ICSI procedure time Some have a delay; pipette continues to move over a small distance. Potential for breakdown, difficult to repair in-house Eppendorf, Narashige


Comments contributed by M. Blayney, A. Burnley, L. Devlin, D. Kastelic, M. van den Berg and B. Woodward.


Microtools

Two types of microtools are used:




  1. 1. Holding pipettes to hold and immobilize the oocyte




    • Outer diameter: 0.080–0.150 mm



    • Inner diameter: 0.018–0.025 mm



    • Fire-polished aperture




  2. 2. Injection pipettes to immobilize, aspirate and inject the sperm cell




    • Outer diameter: 0.0068–0.0078 mm



    • Inner diameter: 0.0048–0.0056 mm



    • Beveled tip, sometimes tipped with a spike.



Both microtools are bent to an angle of approximately 30° at the distal end in order to facilitate horizontal positioning and manipulation adjustment within culture dishes. Aspiration pipettes (of different diameters) may also be used to aspirate anucleate fragments, or to biopsy blastomeres for preimplantation diagnosis. A third type of microtool may be used for piercing or cutting the zona pellucida in assisted zona hatching techniques. Uniform microtool quality is crucial for consistent results, and specifically tooled, sterile, ready-to-use holding and injection pipettes are commercially available. A blunt injection pipette can damage the oocyte by compression, whereas a pipette with too large a diameter will damage the oocyte by injecting too much fluid.



Supplies

Most manufacturers of tissue culture media supply all the components necessary for micromanipulation techniques.



Polyvinyl Pyrrolidine (PVP)

A viscous solution of 10% polyvinyl pyrrolidine can be used to reduce sperm motility prior to immobilization and aspiration into the injection pipette. Experienced operators can carry out the procedure without the use of PVP, but it is helpful in the initial stages of learning and practice. Experimental evidence has shown that PVP can interact with acrosomal and mitochondrial membranes, as well as cause chromatin deterioration after prolonged exposure, and questions have been raised about the wisdom/safety of injecting this artificial agent directly into ooplasm. Although no adverse effects have as yet been reported, PVP should be used cautiously, with attention to the time that the sperm is exposed to the polymer, and with efforts to minimize the amount that is injected into the oocyte cytoplasm.



Hyaluronic Acid (HA)

Hyaluronic acid (HA), a natural component of the cumulus–oocyte complex, can be used as an alternative to PVP. HA has a relatively high negative charge and a high hydration capacity, so that viscous solutions can be prepared which can be used to slow sperm motility for the ICSI procedure; commercial preparations using recombinant HA are available. The motility of spermatozoa in a hyaluronate solution resembles that in the extracellular matrix of mature cumulus cells, and spermatozoa resume normal motility once returned to culture medium. Binding to HA has also been used as a marker for sperm maturity, and this offers an added benefit to its use as an alternative to PVP for moderating sperm motility (van den Bergh et al., 2009); a further advantage is that it degrades to natural sugar molecules that can be readily metabolized by cellular pathways.



Hyaluronidase

This enzyme is used to loosen and disperse cells of the cumulus and corona, prior to their removal from the oocyte by dissection. Preparations of sheep or bovine origin were commonly used in the past, but human recombinant hyaluronidase is now used to minimize risks of disease transmission from animals.



ICSI – Step by Step



Selecting Sperm for Injection

As discussed in Chapter 10, there is evidence that sperm with DNA damage can have an adverse effect on the outcome of ART. Efforts have been made to develop techniques that will enhance sperm preparation methods that can be used to identify and select sperm with lesser levels of chromatin or DNA damage. Technologies that have been applied include magnetic-activated cell sorting (MACS, Said et al., 2008), electrophoretic separation of sperm on the basis of their charge and size (Fleming et al., 2008), binding to HA as an indication of sperm maturity (Huszar et al., 2007), using PICSI (Petri-dish ICSI) dishes containing HA bonded to the Petri dish, assessment of sperm head birefringence (Gianaroli et al., 2008) and the use of high-magnification microscopy (Bartoov et al., 2003).



Magnetic-Activated Cell Sorting (MACS)

This technique aims to reduce the number of spermatozoa with fragmented DNA by eliminating those that are undergoing apoptosis. Such sperm cells may still be motile and have normal morphology. When they reach the terminal phase of apoptosis, phosphatidyl serine is externalized on the external surface of their plasma membrane, and this can be used as a biomarker for apoptosis. Annexin V is a protein that binds phospholipids, but does not pass through the plasma membrane; this molecule can be bound to magnetic microbeads that will covalently bind to phosphatidyl serine on the surface of sperm undergoing apoptosis, and these complexes can be retained in a separation column placed in a magnetic field (MiniMACS Separator). Sperm cells that pass through the column do not express phosphatidyl serine and are therefore identified as non-apoptotic. Studies using this approach for sperm selection indicate that the number of sperm with fragmented DNA can be reduced, with an improvement in acrosome reaction and mitochondrial membrane potential, as well as increase in embryo implantation and pregnancy rates. Stimpfel et al. (2018) carried out a study in couples with teratozoospermia as the main indication for ICSI, using sibling oocytes to compare outcomes. Half of the oocytes were injected with MACS-sorted sperm, and the remainder were injected with sperm prepared by conventional methods. The overall percentage of morphologically normal sperm did not differ significantly between the ICSI and MACS–ICSI procedures, but MACS-selected sperm had more tail abnormalities. Evaluation and comparison of sperm parameters, fertilization and embryo development revealed no significant differences. However, MACS sorting apparently improved the quality of blastocysts in women over the age of 30, but not in younger women. The authors suggest that this may be explained on the basis that oocyte DNA repair capacity decreases with age, and the use of MACS sorting reduces the need for DNA repair in older oocytes. Couples with male infertility due to teratozoospermia in which the female partner is over 30 years of age could theoretically benefit from the use of MACS for sperm selection.



Hyaluronic Acid Binding (HAB)

Hyaluronic acid is a major component of the cumulus complex surrounding human oocytes, and sperm must express HA receptors in order to traverse this complex and reach the oocyte surface. The expression of HA receptors has been reported to be an indication of normal spermatogenesis and sperm maturity, reflecting a number of upstream events that affect DNA integrity and frequency of chromosomal aneuploidies (Mokánszki et al., 2014). A medium rich in HA or Petri dishes with spots of immobilized HA (PICSI dish) can be used for sperm selection. However, a systematic review and meta-analysis of data available up to June 2015 did not confirm an improvement in fertilization and clinical pregnancy rates when HA binding was used as a sperm selection technique in ICSI cycles (Beck-Fruchter et al., 2016).



Digital Holographic Microscopy (DHM)

The sperm cell is almost transparent in conventional bright-field microscopy, as its optical properties differ slightly from that of the surrounding medium. A light beam that passes through a cell undergoes a phase change which depends on the light source and the refractive index of the cell. This ‘phase contrast’ may be observed qualitatively using contrast interference microscopy such as Nomarski Differential Interference (DIC) microscopy and quantitively using digital holography. Digital Holographic Microscopy (DHM) is a noninvasive, label-free, high-resolution phase-contrast imaging technique that can generate automatic three-dimensional images of small cells without mechanical scanning; it has been instrumental in calculating the volume of normal and vacuolated human sperm heads (Coppola et al., 2013, 2017). The volume of a normal sperm head could previously be estimated only by using linear measurements of the head; DHM measures sperm head volume as 8.03 ± 0.75 µm3. DHM can also be used to track sperm in four dimensions in order to compare motility characteristics between normal and anomalous sperm cells (Di Caprio et al., 2014).


DHM may be used in combination with Raman spectroscopy to identify biochemical changes in live human sperm. Current methods of DNA assessment are of limited clinical utility as the technique is invasive and generally based on fluorescence microscopy. Raman spectroscopy is based on the detection of the inelastic scattering of light and provides information on the vibrational states of the illuminated molecules. This technique does not require special dyes or culture medium, it is nondestructive and can be combined with other microscopy techniques such as contrast microscopy or holography. Raman spectroscopy has to date been used successfully to study DNA packaging in human spermatozoa, DNA fragmentation and the identification of X- and Y- bearing spermatozoa in the bovine (Ferrara et al, 2015).


Although these new microscopy techniques require expensive equipment, they continue to provide additional significant information, and may eventually help to identify the ideal spermatozoon for injection into the oocyte.



IMSI (Intracytoplasmic Morphologically Selected Sperm)

Sperm morphology has long been accepted as one of the best indicators for a positive outcome in human fertilization, whether via natural fertilization, IUI, IVF or ICSI. IMSI, a technique using ultra high magnification to select ‘normal sperm,’ has been shown to improve pregnancy rates and decrease abortion rates in some patient categories. This procedure, originally promoted by Bartoov and colleagues (2003), consists of real-time, high magnification, motile sperm organellar morphology examination (MSOME) that uses 24 characteristics to define the normal morphology of seven sperm organelles: acrosome, postacrosomal lamina, neck, mitochondria, tail, outer dense fibers and the nucleus. MSOME is performed with an inverted light microscope equipped with high-power Nomarski optics enhanced by digital imaging that allows the embryologist to magnify sperm up to 6000 times, compared to the traditional 400 times with ICSI. Figure 13.2 shows IMSI photographs of a single normal human spermatozoon and a selection of dysmorphic spermatozoa. Males with severe oligospermia and samples dissected from testicular tissue can potentially benefit from the use of IMSI.





Figure 13.2 IMSI: a single normal human spermatozoon showing true dimensions in micrometers and a selection of dysmorphic spermatozoa. Nomarski optics at ×6000 magnification.



Equipment and Materials for IMSI ––

Sperm observation and selection is carried out using high-magnification objectives, ranging from ×60 in air to ×100 with oil immersion. Hoffman modulation contrast optics are replaced by a system with Nomarski Differential Interference optics, which requires the use of glass-bottomed dishes or slides of 0.17-mm thickness. The optical signal is then enhanced by a video zoom and digital imaging system, giving a final magnification of up to ×10 000. The image is observed, stored and analyzed using specific software supplied by the microscopy company.



Procedure ––

Whereas routine ICSI injection is carried out in plastic dishes with Hoffman optics, IMSI sperm identification and selection is made on glass with Nomarski optics: special glass-bottomed dishes have been designed so that the two procedures may be carried out in the same dish. In this case, the injection procedure is carried out using Nomarski Differential Interference Contrast (DIC) optics. Tissue culture medium, PVP/HA, paraffin oil and microtools remain unchanged. Alternatively, the IMSI selection procedure can be carried out separately on a designated IMSI set up and the selected sperm then transferred to a plastic ICSI dish for microinjection on a routine ICSI micromanipulator.


Hoffman modulation optics can also be adapted/modified to allow selection of sperm at around ×600 magnification without the need for glass-bottomed dishes, but resolution is inferior to the Nomarski system. An excellent and detailed overview of all technical aspects involved in IMSI has been summarized by Vanderzwalmen et al. (2014).


Although IMSI can benefit a targeted group of patients, purchasing the special equipment is expensive, and its application during an ICSI procedure is very time-consuming. Selecting sperm cells on the basis of MSOME criteria can take up to 2 hours (Antinori et al., 2008), and in order to reduce oocyte exposure time, IMSI sperm should be preselected in advance of the injection procedure. Since prolonged sperm handling at 37ºC can also be detrimental, preselection may be carried out at room temperature rather than on a heated stage (Peer et al., 2007). In general, published studies have not shown the use of IMSI to yield significant improvements in fertilization rate; studies show conflicting results, due to differences in inclusion criteria and the numerous confounding variables that arise during IVF/ICSI treatment. It has been suggested that IMSI might be most effective in overcoming ‘late’ paternal effects due to sperm abnormalities at the level of DNA chromatin (Setti et al., 2013).



Oocyte Preparation and Handling

Oocyte retrieval is scheduled after programmed controlled ovarian hyperstimulation (COH), according to protocols described in Chapter 2. Oocyte identification is carried out immediately after follicle aspiration, using a dissecting microscope with heated stage. Take care to maintain stable temperature and pH of the aspirates at all times. At the end of the oocyte retrieval, note quality and assess the maturity of the oocytes, and preincubate them in the controlled atmosphere laboratory IVF incubator at 37°C until preparations are ready for cumulus–corona removal.


The incubation time before removing cumulus–corona cells can vary without significant effects but is usually carried out within 1–4 hours after oocyte retrieval. Studies using Polscope technology to examine the meiotic spindle over time suggest that oocyte aging causes the spindle to become unstable around 12 hours after OCR; injection between 9 and 11 hours post OCR resulted in very poor embryo quality (see Simopoulou et al., 2016 for review). The optimal time for injection appears to be between 37 and 39 hours post hCG, but this may vary in patients with polycystic ovarian syndrome (PCOS). HEPES-buffered media can be used to allow more time for oocyte handling. If non-HEPES media is used, handling outside of the incubator must be kept to an absolute minimum; this option is open only to very experienced personnel.



Cumulus–Corona Removal (Denudation)



  1. 1. Either add one central drop of hyaluronidase solution to the oocyte culture dish at the end of the OCR procedure, or prepare a culture dish containing one central drop of hyaluronidase solution and 5 wash drops of culture medium, covered with an overlay of equilibrated mineral oil (denudation may also be carried out in Nunc four-well dishes). Incubate at 37°C for 30–60 minutes. Note: if HEPES-buffered medium is used, this has been adjusted to pH 7.4 and usually 5 mM bicarbonate. Exposure to a CO2 atmosphere will cause the pH to drop, and therefore culture dishes that contain HEPES-buffered media should be warmed to 37°C in a warming oven, and not in a CO2 incubator.



  2. 2. Prepare a thin glass probe and select denudation pipettes.



  3. 3. Remove the oocyte and hyaluronidase dishes from the incubator. Place one to four oocytes together into the enzyme drop, agitating gently until the cells start to dissociate. Do not leave them in the enzyme preparation for more than 1 minute. Carefully aspirate the oocytes, leaving as much cumulus as possible behind. Wash by transferring them through at least five drops of culture medium, and change to a fine-bore tip for aspiration in order to remove all of the coronal cells.



  4. 4. Assess the quality and maturity of each oocyte under an inverted microscope. Use the glass probe to roll the oocytes around gently in order to identify the polar body, and examine the ooplasm for vacuoles or other abnormalities. Separate metaphase I or germinal vesicle (GV) oocytes from metaphase II oocytes, label them and return to the incubator until ready for the injection procedure.



  5. 5. Examine the oocytes again before starting the injection procedure to see if any more have extruded the first polar body. ICSI is carried out on all morphologically intact oocytes with first polar body extruded. Figure 13.3 illustrates different stages of egg maturity that are revealed after hyaluronidase treatment and corona dissection.





Figure 13.3 Variations in egg maturity found after hyalase treatment and corona dissection. (a) germinal vesicle; (b) metaphase I; (c) metaphase II.



Preparation for Injection



  1. 1. Prepare injection dishes with 4–8 droplets of 2–5 μL of HEPES-buffered culture medium for each individual oocyte, and a 5-μL droplet of PVP or HA for the sperm. The droplets can be arranged in a circle with the sperm selection drop in the center, or in parallel groups, but must be positioned so that they are not too close to the edge of the dish, where manipulation will be difficult. The oocyte droplets should not be too close to the sperm droplet, in order to avoid mixing; use an arrangement that allows quick and easy distinction between sperm and oocyte droplets, with numbers etched on the bottom of the dish if desired. Small volumes of media evaporate very quickly, and they should be covered immediately with a layer of oil. Equilibrate the dishes in the incubator for at least 20–30 minutes, and keep them in the incubator until you are ready to begin the procedure. If Falcon 1006 dishes are being used with HEPES-buffered medium, the lids must be tightly fixed if they are to be equilibrated in a CO2 incubator. Dishes with HEPES medium and no lids should be warmed to 37°C without CO2 atmosphere.



  2. 2. A prepared pre-equilibrated traditional culture dish should be available, to transfer and further culture the oocytes after injection.



Micromanipulator



  1. 1. Make sure that the microscope heating stage is at a temperature that will maintain droplet temperature in the dishes at 37°C, ensure that all controls are set to neutral and can be comfortably operated and that you are confident that all parts function smoothly before you begin. It is essential to check that you can smoothly carry out very small movements. This involves not only the equipment itself, but its position on the bench in relation to your (comfortable) seating position.



  2. 2. Insert holding and injection pipettes into the pipette holders, tighten well and if an oil-filled system is being used, make sure that there are no air bubbles in the tubing system. Bubbles interfere with sensitivity when attempting to control movement with fine precision.



  3. 3. Align the pipettes so that the working tips are parallel to the microscope stage. First align the holding pipette under low magnification, then align the injection pipette, again under low magnification. Check the position of both under high magnification. It is important to begin with pipettes in accurate alignment: both working tips must be sharply in focus. If a part of the length is out of focus, the pipette is probably not parallel to the stage, but pointing upwards or downwards.



  4. 4. Adjust the injection controls: if using an oil system, the oil should just reach the distal end of the pipette; do not try to fill the needle with oil, as this will work only if you leave a tiny 5-mm gap of air between the oil and the medium. Briefly touch the tips of both pipettes in oil, and then in medium, so that the ends fill by capillary action. Apply positive pressure on the injector to hold the oil bubble at this point – the bubble of oil behind the medium acts as a buffer and should be kept in this area throughout the injection procedure. Moving the bubble further up the needle increases suction power and reduces sensitivity; moving it nearer the tip has the opposite effect and also creates a risk of injecting oil into the oocyte. The injection dish is still in the incubator, so you should be using a ‘blank’ dish to make these adjustments.



Transfer of Gametes to the Injection Dish



  1. 1. Add a small aliquot of sperm suspension (0.3–0.5 mL, depending on the concentration of prepared sperm) to the edge of the central PVP/HA droplet. The viscous solution should facilitate sperm handling by slowing down their motility and also prevents the sperm cells from sticking to the injection pipette during the procedure. Be careful of sperm density: too many sperm will make selection and immobilization more difficult.



  2. 2. After the sperm droplet has been examined for the presence of debris or any other factors that might cause technical difficulties, examine all the denuded oocytes again for the presence of a first polar body; wash them with HEPES-buffered medium and transfer one oocyte into each oocyte droplet on the injection dish, taking care to avoid too much handling or cooling of the oocytes. Keep the oocytes in the incubator until you are confident that the injection procedure can proceed smoothly. Until sufficient experience of the procedure has been gained, it may be advisable to keep sperm and oocyte dishes separate, avoiding overexposure of the oocytes while selecting and immobilizing sperm.



  3. 3. Place the injection dish with central sperm droplet on the microscope stage. Using the coarse controls of the manipulator, lower the injection pipette into the drop.



Sperm Selection and Immobilization



  1. 1. Select sperm that appear morphologically normal. Sperm can be selected and stored in the PVP drop for a limited period of time (be aware that prolonged exposure to PVP can cause damage to sperm membranes) before starting the injection procedure; this is an advantage in cases of extreme cryptozoospermia, and reduces oocyte exposure time. If medium containing HA is being used for immobilization, the sperm become rigid and stick to the dish after approximately 30 minutes.



  2. 2. Immobilize motile spermatozoa by crushing their tails: select the sperm to be aspirated, and lower the tip of the injection needle onto the midpiece of the sperm, striking down and across, and crushing the tail against the bottom of the dish. This ‘tail crushing’ impairs motility and destabilizes the cell membrane; the latter may be required for sperm head decondensation. If the resulting sperm has a ‘bent’ tail, it will be difficult to aspirate into the needle, and will stick inside it. When this happens, abandon that sperm and repeat the procedure with another sperm. Do not strike too hard, or the sperm will stick to the bottom of the dish, also making aspiration into the needle difficult. After some practice, sperm immobilization in routine ICSI cases can be carried out quite quickly. If the preparation contains only a few sperm with barely recognizable movement and a large amount of debris, this part of the procedure can be very tedious and require great patience!



  3. 3. Aspirate the selected immobilized sperm into the injection pipette. Sperm were traditionally aspirated into the pipette tail-first, but they can be aspirated head-first (Woodward et al., 2008a). Position the sperm approximately 20 µm from the tip.



  4. 4. Lift the injection needle slightly, and move the microscope stage so that the injection pipette is positioned in the first oocyte drop. If the sperm moves up the pipette (due to the difference in density between culture medium and PVP), bring it back near the tip before beginning the injection procedure.



Injection Procedure



  1. 1. Lower the holding pipette into the first oocyte droplet, and position it adjacent to the cell. Using both microtools, slowly rotate the oocyte to locate the polar body. Aspirate gently so that the cell attaches to the pipette. The pressure should be great enough to hold the oocyte in place, but not so strong that it causes the oolemma to bulge outwards.



  2. 2. Positioning the polar body at 6 or 12 o’clock in order to minimize the possibility of damaging the meiotic spindle was thought to be important, but later evidence using polarized microscopy to visualize the spindle itself suggests that polar body positioning is of less benefit than minimizing the duration of the ICSI procedure (Woodward et al., 2008b).



  3. 3. Move the injection pipette close to the oocyte, and check that it is in the same plane as the right outer border of the oolemma on the equatorial plane at the 3 o’clock position. Check that the sperm can be moved smoothly within the injection needle, and position it near the beveled tip.



  4. 4. Advance the pipette through the zona pellucida until the tip almost touches the oocyte membrane at the 9 o’clock position (Figure 13.4). If the pipette is in the wrong plane, entry into the cell will be difficult. The membrane may rupture spontaneously, or may require negative pressure, sucking the membrane into the pipette before expelling the sperm. When it breaks, there will be a sudden flux of cytoplasm into the pipette. Inject the sperm slowly into the oocyte with a minimal amount of fluid (1–2 picoliters). The sperm should be ejected past the tip of the pipette, to ensure a tight insertion among the organelles, which will hold it in place while the pipette is withdrawn. Some surplus medium may be re-aspirated to reduce the size of the breach created during perforation. If the plasma membrane is elastic and difficult to break, it may be necessary to withdraw the pipette from the first membrane invagination and slowly repeat the procedure. Oocytes do vary in their response to injection, and it is important to remain flexible and adapt technique accordingly.



  5. 5. Withdraw the injection pipette, and examine the breach area. The membrane should be funnel-shaped, pointing in toward the center. If the border of the oolemma is everted, cytoplasm may leak out, and the oocyte may subsequently cytolyze. Release the oocyte from the holding pipette.



  6. 6. Repeat the sperm aspiration and injection until all the selected metaphase II oocytes have been injected.



  7. 7. Wash all the oocytes in culture medium, transfer to the prepared, warmed culture dish and incubate overnight.






(a) Metaphase II oocyte with injection needle in position prior to injection.





(b) Injection needle within the cytoplasm, prior to release of sperm.





(c) Post-injection illustrating the typical track left following withdrawal of the needle.





(d) Post-injection, oocyte with a very elastic membrane.


With thanks to Agnese Fiorentino.


Figure 13.4 ICSI.




Injection Procedure: Important Points




  1. 1. All conditions must be stable: temperature, pH, equipment properly set up, adjusted, aligned, and checked for leaks and air bubbles. Check everything, including secure and comfortable operating position, before you begin.



  2. 2. Correct immobilization of sperm.



  3. 3. Advance far enough into the ooplasm with the injection pipette.



  4. 4. Ensure that the plasma membrane is broken. (Immediate membrane rupture after introducing the injection needle results in a lower probability of fertilization.)



  5. 5. Inject a minimal volume.



  6. 6. If the sperm comes out of the ooplasm into the perivitelline space, reinject.



Assessment of Fertilization and Cleavage

Around 16 to 18 hours after injection, assess the number and morphology of pronuclei through an inverted microscope. Polar bodies can also be counted, with reference to digynic zygotes or activated eggs; polar bodies may fragment, even in normal monospermic fertilization. Rapid cleavage (20–26 hours post-injection) can occur in ICSI zygotes (see also ‘silent fertilization’ in Chapter 5: ‘Causes of Early Embryo Arrest’).


Evaluate normally fertilized, cleaved embryos after a further 24 (or 48) hours of culture, and continue culture for transfer or freezing at cleavage or blastocyst stage according to laboratory protocols.



No Fertilization after ICSI

Complete failure of fertilization is rare after ICSI (reported as 1–3% of cycles), but can occur as a result of sperm defects, oocyte defects or technical problems.


Sperm defects include:




  • lack of motile sperm



  • failed sperm head decondensation



  • premature sperm chromatin condensation



  • sperm aster defects



  • round-headed sperm (globozoospermia)



  • severe oligospermia.


Fertilization rates using epididymal and testicular sperm are generally equivalent to those for ejaculated sperm, but the use of immature sperm cells results in a dramatic decrease in fertilization and pregnancy rates.


Oocyte defects include:




  • low oocyte numbers



  • abnormal oocyte morphology



  • spindle defects



  • failure of oocyte activation



  • fragile oocytes which are easily damaged by the trauma of the injection.


Unfertilized oocytes can show abnormal spindle and interphase microtubules, suggesting that deficiencies in ooplasmic and nuclear components may be responsible for failed fertilization.


Failure can also be due to technical problems involving incorrect injection procedure or highly elastic plasma membranes that impede complete rupture of the oocyte membrane, so that the sperm cell fails to be placed into the oocyte cytoplasm. Figure 13.5 shows the variations that can be seen on Day 1 after ICSI.


Sep 17, 2020 | Posted by in OBSTETRICS | Comments Off on Chapter 13 – Micromanipulation Techniques
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