MICROMANIPULATION OF OVA AND BLASTOCYSTS (TABLES 16.1 AND 16.2)
Various methods have been employed for embryo sexing, such as separation on the basis of sexual dimorphism, detection of sex chromatin mass, fluorescence in situ hybridization, detection of the HY antigen and use of a Y-specific DNA probe. Several processes are involved in nuclear transfer including use of a particle gun, sperm-mediated gene transfer, injection of the pronuclei, retroviral-mediated gene transfer, injection of the oocyte cytoplasm and germinal vesicle gene injection.
Zona pellucida
The zona pellucida, an extracellular lipoprotein matrix, serves several functions during the induction of the sperm acrosome reaction, which is stimulated by exposure of sperm to intact or digested zonae, during cleavage, and hatching of the blastocyst. Release (hatching) of the blastocyst from the zona pellucida occurs in the uterus 6 days postovulation (Figure 16.1). Several techniques have been used for embryo micromanipulation (Figure 16.2). The success of the techniques depends on the use of agar gel to seal incisions made in the zona pellucida during micromanipulation. The agar protects the blastomeres from damage by uterine secretions and leukocytes until the embryo has developed sufficiently to survive in utero without a zona.
Zona drilling/cracking
Zona drilling may be complete or partial zona drilling (PZD) (Figure 16.3). Zona drilling is accomplished mechanically or by local application of a zona solvent such as acid Tyrode’s or alpha-chymotrypsin solution using a microneedle. In the case of PZD the zona is mechanically torn with a glass needle or cracked by piercing it with a glass holding pipette. PZD is usually followed by microinsemination (Figure 16.4). For ‘cracking’ of the blastocyst, two fine glass hooks are controlled by a micromanipulator. In blastomere transfer, part of the cell, specifically the nuclei, or whole cells are transferred. Fusion of cytoblast/ karyoplasms with host cells is inhibited by electrofusion. For blastocyst biopsy one or two blastomeres are removed (for preimplantation diagnosis of sex or genetic anomalies) allowing the remaining embryos to develop.
Bisection of blastocysts
Six microinstruments are placed in the optical field of an inverted microscope. Two micropipettes controlled by type-B micromanipulators hold the blastocyst and the empty zona pellucida by negative pressure. Instruments with a sharp tip positioned in front of the blastocyst are controlled by a type-A micromanipulator. The micropipette on the left and the microscalpel on the right are controlled by a type-B micromanipulator. Two sharp microneedles cut the zona pellucida over as short a distance as possible along the middle. The blastocyst is subsequently rotated through 90° with the two microneedles to show the slit. The micropipette is introduced through the slit inside the zona, while a small volume of medium is injected to expel the embryo. Bisection of the blastocyst is achieved using the microscalpel along the sagittal plane. Using the suction of the micropipette, each ‘half’ embryo is put back into an empty zona pellucida. ‘Half’ (demi) embryos are then incubated at 37°C for 2h. They reconstitute the blastocele, and it is possible to distinguish again the inner cell mass in each ‘half’ blastocyst. The cells destroyed during the bisection adhere to the outer surface of the ‘half’ blastocyst.
CRYOPRESERVATION OF GAMETES AND EMBRYOS (FIGURE 16.5)
The main advantage of cryopreservation of embryos, instead of just the sperm or oocytes, is that the embryo contains the complete genome, i.e. the full quota of chromosomes for the individual, and it can be transferred to a foster mother of known or unknown genetic background without the risk of genetic change. Since the pioneering efforts of Audrey Smith in 1952 concerning the effect of low temperature on further development of mammalian ova, much progress in embryo cryopreservation has been made. Cryopreservation of embryos of different mammalian species has been attempted with variable degrees of success.
Principles of cryobiology
The biophysical principles that apply to cryopreservation of living cells and tissues also apply to cryopreservation of embryos. Embryos may be damaged during cryopreservation and/or thawing by either the formation of large intracellular ice crystals or the increased intracellular concentration of solutes and the accompanying changes that result from dehydration of the cells during cryopreservation (solution effects). Whereas fast freezing minimizes damage from solution effects, it leads to the formation of large ice crystals that cause severe mechanical damage. On the other hand, while slow freezing prevents large ice crystal formation, it leads to increased damage from solution effects. Therefore, the optimal freezing rate for a given tissue depends on its relative tolerance to damage from ice crystals and toxicity from solution effects.
The critical range of temperatures at which slow rates are necessary for optimal survival during cooling are from -70°C to -20°C. Human embryos can be preserved for prolonged periods in a state of suspended animation if they are able to withstand cryopreservation to temperatures at which no further biological activity occurs. Liquid nitrogen at -196°C satisfies this condition.
Cryopreservation of embryos (Tables 16.3-16.5)
Various techniques have been used for cryopreservation and thawing. Embryos selected for cryopreservation should be of the highest quality and at the correct stage of cleavage. They are handled with sterile techniques using a dissecting microscope. Embryos are transferred to sterile, freshly prepared culture media for microscopic classification and storage until use. If embryos are stored for longer than 2 h before transfer, they should be transferred into fresh medium every 2 h. The embryos are aspirated into micropipettes with a small volume of medium (less than 0.2 ml) to prevent contamination of the fresh medium. The embryos are handled gently to avoid any physical damage. Manipulation and evaluation are accomplished as quickly as possible to enable the embryos to be returned to a stable culture environment. To gain experience with handling embryos, operators are trained to use commercially available micropipettes to pick up Sephadix® particles with a diameter similar to that of mammalian eggs. Pieces of debris, unfertilized ova or degenerating ova can also be used for practice.
Figure 16.1 Blastocyst expansion, hatching and implantation.The morula enters the uterus on day 3, grows and cavitates to form a blastocyst. (a) Blastocyst, hollow ball of cells surrounded by zona pellucida (ZP). (b) Blastocyst hatching: blastocyst expands and ruptures ZP, emerging as a dumb-bell shape. (c) Early implantation in the endometrium. BV, blood vessel; C, cytotrophoblast; E, embryoblast; Ep, epithelium; En, endometrium; ST, syncytiotrophoblast;T, trophoblast; UG, uterine gland
Figure 16.2 (a)-(d) Micromanipulation of the ovum to remove the zona pellucida to be used for biological assay. (e) and (f) Insertion of single blastomere into the zona pellucida
Post-thaw embryo survival
Several maternal, technical and operational factors influence the survival rate of embryos:
(1) Physiological and biophysical characteristics and developmental stage of embryos;
(2) Time interval and in vitro treatment from embryo collection to initiation of cryopreservation;
(3) Type of computerized freezers and program of cryopreservation;
(4) Osmotic shock during various stages of cryopreservation;
(5) Exposure of embryos to excessive light during microscopic examination;
(6) Osmotic and colloid osmotic pressures of fluids, prepared media and cryoprotectants;
(7) Nature and extent of ‘seeding’ and ‘plunging’;
(8) Microbiological contaminants in glassware, freezer and storage.
Figure 16.3 Partial dissection of zona pellucida
Semen cryopreservation
Cryopreservation is a useful and often necessary process for semen storage. During cryopreservation, sperm are kept in a state of suspended animation within a deep-frozen medium (liquid nitrogen). Azoospermic patients have extremely abnormal sperm quality, and may be infected with a sexually transmissible disease (STD), such as acquired immune deficiency syndrome (AIDS). Donor (homologous semen) sperm is required for procreation and must be cryopreserved and quarantined for at least 6 months before use to prevent STD infections. If the husband is unable to provide a semen sample on demand, or is unable to provide a semen sample in the future (as in cases of chemotherapy), the husband’s ejaculate (autologous semen) has to be cryopreserved at the time of insemination; the cryopreserved sample is then thawed and processed for use.
Figure 16.4 Sperm microinjection/microinsemination. (a) Microinsemination sperm transfer (MIST): egg held by holding pipette (HP), while sperm are injected into perivitelline space (PVS) beneath zona pellucida (ZP) using fine injecting needle (I). (b) Sperm microinjection directly into cytoplasm of oocyte. One sperm is injected directly in ooplasm. (c) Zona drilling: hole in zona drilled using acid Tyrode’s medium, through which sperm introduced into PVS. (d) Microinsemination: zona opening or zona cutting using hooked needles and partial zona dissection (PZD) where opening is made with microneedle insemination of oocyte
Figure 16.5 Possible options for restoring fertility using cryopreserved immature and mature male germ lines. AI, artificial insemination; IVF, in vitro fertilization; IVFC, in vitro fertilization and culture; IVC, in vitro culture; ICSI, intracytoplasmic sperm injection
Several extending media are used for semen cryopreservation and perform different functions, including providing nutrients as a source of energy, protecting against the harmful effect of rapid cooling, providing a buffer to prevent harmful shifts in pH as lactic acid is formed, maintaining the proper osmotic pressure and electrolyte balance, inhibiting bacterial growth and protecting the sperm cells during freezing. A simple carbohydrate, such as glucose, usually is added as a source of energy for the sperm. Both egg yolk and milk are use to protect against cold shock of the sperm cells as they are cooled from body temperature to 5°C. These substances also contain nutrients that can be used by sperm. A variety of buffers may be used to maintain a nearly neutral pH and an osmotic pressure of approximately 300mmol/l, which is equivalent to that of semen and blood plasma.
Sperm after cryopreservation
The sperm plasma membrane contains high levels of unsaturated fatty acids and is therefore particularly susceptible to peroxidative damage with subsequent loss of membrane integrity and impaired cell function of sperm. After storage of the frozen semen for 1 month and 2 years the percentage of damaged sperm is almost the same. There is no significant difference in the fertilizability of semen. In bull sperm after thawing, selenium and vitamin E stimulate sperm motility and viability. In medium containing 0.5 mg vitamin E/ml diluted semen, the fertility of thawed sperm is 5% higher. The addition of different antioxidants to diluents at storage/cryopreservation is recommended to improve the quality of semen used for artificial insemination (AI).
Programmable freezers
Electronically programmed machines are used to monitor the temperature over the critical phase of the cooling curve before the vials containing embryos are plunged into liquid nitrogen. Several types are completely self-contained and capable of obtaining controlled rates of cryopreservation with or without liquid nitrogen and without mechanical refrigeration devices or the use of conventional refrigerants. The three major systems are freezers with a cylindrical chamber with circulating air cooled by microprocessor controlled thermoelectric cells, freezers operated with liquid nitrogen and freezers operated with alcohol or other agents.
Table 16.3 Precooling, cooling, seeding, plunging and storage and thawing of embryos
|
Process |
Techniques employed |
|
Collection of embryos |
Selection and superovulation of donor, insemination during estrus Collection of embryos from female reproductive tract or ovaries (surgical, non-surgical or postmortem) Washing of embryos in sterile culture media Microscopic evaluation and classification of embryos |
|
Cryoprotectant solutions |
Gradual step-wise concentration of cryoprotectant (DMSO, glycerol or ready-made commercially available cryoprotectant) Small volume is freshly prepared Solutions to which serum is added are not stored more than 3-5 days, because protein denaturation occurs even at optimal temperatures Solutions are deep refrigerated or frozen until use |
|
Precooling preparation |
Embryos transferred in serial concentrations of cryoprotectant Straws attached to syringe using rubber adapter to aspirate medium or air bubbles and embryo Straws and cane labeled for future identification Straws heated or filled with phosphate-buffered saline Straws dipped in blue or red PVC at both ends |
|
Cooling procedures |
Slow cooling rate ranges from 0.5 to 1.6°C/min Rapid cooling rate ranges from 17 to 30°C/min Cooling rate 1°C/min from ambient temperature to - 7°C Cooling rate 0.3°C/min to - 35°C Cooling rate 0.1°C/min to - 38°C |
|
Freezing, seeding and plunging |
Straws placed in freezer at - 6°C and maintained for 10 min Forceps cooled in liquid nitrogen Straws grasped near embryos with cooled forceps until ice crystals form Seeded straw placed into programmable freezer and cooling regimen applied Thermos flask filled with liquid nitrogen Cane containing frozen embryos removed from freezer and plunged into liquid nitrogen Straws loaded in aluminum canes and stored in liquid nitrogen container at - 196°C |
|
Thawing |
Thawing ranges from 20°C/min in slow warming to 360-500°C/min for the rapid thawing; optimal temperature for cryopreservant thawing ranges between 20 and 37°C Temperature of water bath adjusted to 37°C Color-marked canes identified and straws removed from cane to small canister containing liquid nitrogen; labels are checked before removing from liquid nitrogen Straws are held by neck, placed in 37°C water bath and removed when ice melts Embryos remain on bottom of vial and are observed under a microscope, counted and removed with a micropipette to avoid the occasional loss of embryos experienced by washing embryos out of the straw Straws are thawed for 4 s in a 37°C water bath and removed when ice melts Water is wiped from straws |
|
Cryoprotectant removal |
Heat seal or PVC plug cut from tips of straws Embryos washed through drops of serial dilutions of cryoprotectant mixture in sterile Petri dish (35 mm diameter) Embryos examined using a stereoscope to evaluate their quality |
DMSO, dimethyl sulfoxide; PVC, polyvinyl chloride
Table 16.4 Morphological classification of embryos before and after cryopreservation and thawing
|
Parameter |
Classification |
|
Stage of embryonic development |
Unfertilized (UFO) 2-12-cell Early morula Morula Early blastocyst Blastocyst Expanded blastocyst Hatched blastocyst Expanding hatched blastocyst |
|
Criteria for classification of embryos |
Compactness of blastomeres Regularity in shape of embryo Variation in cell size Color and texture of the cytoplasm Presence of vesicles Presence of extruded cells Diameter Regularity of the zona pellucida Presence of cellular debris |
|
Quality of embryo |
Excellent: perfect embryo for its stage. Blastomeres are of similar size with even color and texture; they are neither very light nor very dark. Cytoplasm is not granular or unevenly distributed and contains some moderate-sized vesicles. Perivitelline space empty and of regular diameter;zona pellucida even and neither wrinkled nor collapsed |
|
Good: trivial imperfections such as an oval zona, a few small excluded blastomeres and slight asymmetry |
|
|
Fair: definite but not severe problems such as moderate numbers of excluded blastomeres, small size and small amounts of degeneration |
|
|
Poor: partly degenerated, vesiculated cells, greatly varying cell size, very small, and/or similar problems |
|
|
Very poor: severely degenerated, probably not worth transferring, unfertilized,zona only,ghost-like, 3-cell, debris, bacteriological contamination |
|
|
Artifacts |
Air bubble, debris, empty zona pellucida, denuded oviductal epithelium |
ASSISTED REPRODUCTIVE TECHNOLOGY (TABLES 16.6 AND 16.7; FIGURE 16.6)
During the recent past there have been dramatic changes in the field of male infertility. The introduction of intracytoplasmic sperm injection (ICSI) and specialized sperm retrieval procedures has made it possible to circumvent some of the most severe forms of male infertility. If at least one viable sperm cell is available, the pregnancy rate with ICSI depends on the age of the woman and the number of oocytes retrieved.Wood and associates (1993) at the Liverpool Women’s Hospital, UK, reported that motile epididymal sperm retain their fertilizing capacity after cryopreservation. The data on testicular sperm suggest that, although cryopreservation may reduce the fertilizing capacity of testicular sperm, there is no decrease in pregnancy rate.
Embryo donation
The American Society for Reproductive Medicine (ASRM) provides recommendations for the evaluation of potential sperm, oocyte and embryo donors, incorporating recent information about optimal screening and testing for sexually transmitted infections (STIs), genetic diseases and psychological assessments. This represents an effort to make the screening guidelines for embryos and gametes more consistent and incorporates recent information from the Centers for Disease Control (CDC), Food and Drug Administration (FDA) and American Association for Tissue Banks (AATB). The term ‘screening’ refers to specific historical factors that place an individual at high risk for a given disease such as human immunodeficiency virus (HIV) and transmissible spongiform encephalopathy (TSE) or Creutzfeldt-Jakob disease (CJD). ‘Testing’ refers to specific laboratory studies such as serological tests.
Table 16.7 Assisted reproductive technology/andrology (ARTA) and in vitro semen manipulation
|
Method |
Techniques |
|
Assisted reproductive technology/ andrology (ARTA) |
IVFET (in vitro fertilization/embryo transfer) GIFT (gamete intrafallopian transfer) PROST (pronuclear-stage tubal transfer) ZIFT (zygote intrafallopian transfer) TEST (tubal embryo-stage transfer (embryos in premorula cleavage stages)) Embryo bank (cryopreservation of oocytes/morulae) |
|
Zona drilling |
Mechanical or by local application of a zona solvent such as acid Tyrode’s or alpha-chymotrypsin solution using a microneedle |
|
Partial zona drilling (PZD) |
Zona is mechanically torn with a glass needle or cracked by piercing on a glass holding pipette |
|
Subzonal injection (SUZI) |
Injection of one or more sperm into perivitelline space |
|
Intracytoplasmic sperm injection (ICSI) |
Injection of immotile live sperm directly into vitellus |
|
Semen manipulation in vitro |
Swim-up/swim-down Use of semen additives In vitro capacitation In vitro hyperactivation Separation of X and Y sperm Cryopreservation/semen banks |
ASRM also established guidelines for assisted reproductive technology (ART) clinics wishing to offer embryo donation, guidelines for couples who wish to donate an embryo, guidelines for potential recipients, record keeping and protection of confidentiality. Genetic screening is established for various ethnic groups including Ashkenazi Jews (for Tay- Sachs disease, Canovan disease), African-Americans (for sickle cell anemia); Mediterranean people (for P-thalassemia); south-east Asians and Chinese (for a-thalassemia) and Caucasians with European descent (for cystic fibrosis).
Intrauterine insemination versus in vitro fertilization/intracytoplasmic sperm injection
A variety of cases of infertility are treated with intrauterine insemination (IUI), with or without controlled ovarian hyperstimulation (COH), rather than with ICSI and in vitro fertilization (IVF). IUI is efficient and cost-effective for the treatment of unexplained and not severe male factor infertility. IUI is also applied to specific types of female infertility such as oligo-inoculators, cervical factor and corrected minimal tubal obstruction. The IUI outcome, in terms of clinical pregnancy rate, is influenced by various factors including the results of COH, timing/number of insemination(s), duration/ cause of infertility, age of the woman, number of follicles, endometrial thickness or organization at the time of ovulation. Of parameters related to the male partner, the most important determinant of pregnancy outcome is sperm progressive motility.
However, samples with an acceptable number of motile sperm can be processed efficiently.
Figure 16.6 At the time of fertilization, the morning following the day of injection of oocytes they typically appear: (a) unfertilized, (b) monopronucleate, (c) fertilized, (d) triploid, (e) degenerate. From Fleming and King (2003), with permission
Smoking and IVF/ICSI
Cigarette smoking by men and/or their female partners reduces fecundity and the success rate of IVF and ICSI, owing to the harmful effect of benzopyrene and nicotine, noxious metabolites of cigarette smoke. This is associated with a reduction in sperm motility and/or morphology as well as alteration in sperm membrane function, with a subsequent decrease in the ability to undergo capacitation and oocyte penetration. Apart from putative adverse effects during fertilization, altered DNA in sperm might hamper development of the embryo (Zitzmann et al., 2003).
ICSI and calcium ionophore oocyte activation
Severe oligoasthenoteratozoospermia is usually treated with ICSI, since such patients have no or very low fertilization rates using conventional IVF. Couples with unexplained infertility after superovulation and IUI undergoing IVF have an 11-23% chance of fertilization failure that can be easily overcome by using ICSI. The fertilization rate after ICSI is some 70%. Total fertilization failure after ICSI can be due to various mechanisms related to the oocytes or qualitative or quantitative low semen characteristics, such as total immotile or rounded-head sperm. Calcium ionophore oocyte activation seems to be a useful method in cases of repeated failed fertilization after ICSI (Eldar-Geva et al, 2003).
Anabolic steroid-induced azoospermia
Power lifters, body-builders and other related professional athletes are known for anabolic steroid abuse. Anabolic steroids increase lean muscle mass while suppressing the hypothalamic-pituitary-gonadal axis. This is associated with hypogonadotropic hypogonadism and oligospermia or azoospermia. Menon (2003) was able to treat this condition with the combined administration of human chorionic gonadotropin (hCG) and human menopausal gonadotropin (hMG).
UROLOGICAL SURGERY
Care must be taken during andrological surgery, including use of aseptic techniques, meticulous hemostasis, counting of sponges before wound closure and follow-up care.
Pathophysiology, risk factors, complications and related management of urological surgery
Damage to the urinary sphincter during transurethral resection of the prostate can cause incontinence which may also be due to underlying neuro-urological problems undiagnosed before the procedure. A thorough history and physical examination is imperative before assuming that lower urinary symptoms are owing solely to bladder outlet obstruction as opposed to bladder dysfunction. If bladder outlet obstruction necessitates surgical intervention, resection of prostatic tissues is limited to tissue proximal to the verumon- tanum, using video-cystoscopy/photography of the external urinary sphincter. The introduction of a-blockers has radically changed the management of benign prostatic hyperplasia (BPH). Patients who fail medical therapy require transurethral resection of the prostate.
Scrotal pain in the prepubertal male is testicular torsion until ‘proven otherwise’. Testicular torsion occurs at any age, and must be included in the differential diagnosis of any male with acute onset of scrotal discomfort. If misdiagnosised as epididymitis the patient is given a prescription for antibiotics and anti-inflammatories and sent home. An anterior lie of the epididymis and absence of the cremasteric reflex on the symptomatic side can be signs of testicular torsion. The diagnosis of acute testicular pain is aided by either Doppler ultrasonography or nuclear scrotal scintigraphy.
Hematoma formation, the most common surgical complication after vasectomy, occurs as a result of vasospasm during the procedure with delayed bleeding after wound closure, or suture material or surgical clips which fall off. A hematoma can become infected resulting in loss of the testis. No-scalpel vasectomy can reduce the incidence of complications.
Epididymitis/orchitis can occur even with antibiotic prophylaxis. Patients should be informed that the effectiveness of the procedure must be demonstrated by semen analysis before the cessation of contraception.
Both radical and partial nephrectomy for renal cell cancer are performed laparoscopically. Benefits to patients are shorter hospital stay, less pain and faster recovery to baseline. Patient candidates for nephrectomy are given the option of having the procedure performed laparoscopically. Furthermore, informed consent should consider all possible treatment options, including open/laparoscopic radical nephrectomy, partial nephrectomy or cryosurgical ablation of the lesion. Patients should be informed of potential injury to the bowel, blood vessels and other adjacent structures during these procedures. A second additional procedure (repair of bowel injury) may be necessary if patients are willing to undergo such a procedure if needed. Symptoms of hyponatremia can start as mental confusion, or muscle cramps, and progress to convulsions, then coma or death.
The syndrome can be avoided by limiting resection time to less than 1.5 h, since the volume of glycine irrigant absorbed is proportional to the length of the procedure. Transurethral resection syndrome must be suspected in a patient who experiences hemodynamic changes, including hypertension and bradycardia. These may be the only signs of hyponatremia in the anesthetized patient. In more severely affected patients, seizures can occur. The anesthesiologist must note these changes and communicate with the surgeon. Awareness of the symptoms of hyponatremia and aggressive therapy once diagnosis is suspected are critical to optimal management.
Radical prostatectomy, the ‘gold standard’ for treatment of localized prostate cancer, is associated with significant intra-/postoperative morbidity. Large- volume blood loss occurring in the perioperative period requires blood transfusion. Injury to the rectum is one of most feared complications during radical prostatectomy. While it is possible to perform primary repair of an injury to the rectum, some patients may require a temporary colostomy. Appropriate preoperative bowel preparation (mechanical and antibiotic) allows for primary repair of a rectal injury, thereby avoiding colostomy.
Long-term complications associated with radical prostatectomy include incontinence, stricture formation at the bladder neck and impotence. These complications can be managed medically or may require surgery. Insertion of radioactive seeds within the prostatic tissue (brachytherapy) has gained significant popularity. Improvements in imaging technology have enabled mapping of the prostate and precise radioactive seed placement. This procedure is performed on an ambulatory basis and does not require prolonged catheterization, unlike radical prostatectomy. Brachytherapy is efficacious for appropriately selected patients. Morbidity following brachytherapy includes urinary incontinence, urethral stricture, impotence and recto-prostatic fistula formation.
Cryosurgery is performed for both primary and recurrent disease. With the routine use of urethral warming devices and rigorous temperature monitoring within the prostate and at the Denonvilliers’ fascia, the extent of the tissue freezing can be precisely controlled. Technical advances in cryosurgery are of significant potential for both short- and longterm morbidity, including urethral sloughing, irrigative voiding symptoms and recto-prostatic fistula formation.
Prostate-specific antigen (PSA) testing, digital rectal examination and improvements in biopsy techniques have enabled prostate cancer to be diagnosed in more males at an earlier and, therefore, presumably curable stage. All men over 50 years of age should be screened for prostate cancer. Screening should be performed annually using PSA and digital rectal examination. For African-American males or those with a family history of prostate cancer, screening should begin even earlier (40 years). Screening should also be undertaken when various complications occur, including urethral tears, creation of false passages into the urethra, urinary tract infections, bleeding, undermining of the bladder neck/bladder perforation during manipulation of the penis or urethra or when the site of urethral perforation is the bulbar urethra, because of the curvaceous course of the urethra at this point. In bladder neck perforation of the urethra at the bulb, use of internal, self-retaining ureteral stents improves patient comfort/reduces the likelihood of stent migration. Because of the nature of these devices, one can lose track of them until complications arise. The most common complications are bacterial or fungal urinary tract infections. Encrustation of the stent from prolonged retention, making endoscopic removal of the stent impossible, is another complication. One preventive measure is to keep a log of all stents placed, with the date of stent removal. Common causes of complications are compression or occlusion of the vasculature of the spermatic cord, causing closure of the internal inguinal ring; direct clamping or cutting, damaging the cord structures/injuring the testicular artery; painful, tender/swollen testis; herniorrhaphy causing protracted discomfort, ischemic injury or testicular dysfunction; damage as a result of the mesh used in inguinal herniorraphy of the spermatic cord, the mesh eroding into the vas deferens or causing fibrotic reaction/vasal obstruction; and vas deferens injured during dissection of the hernia sac from cord structures or during sac ligation (Meray et al, 2002).
Percutaneous nephrolithotripsy is a ‘minimally invasive’ procedure associated with reduced morbidity, compared with open procedures. As guide wires or needles are blindly introduced via the flank into the kidney, perforation of the kidneys, arteries, veins and even bowel can occur. In the event of complications, a nephrectomy or other procedure to repair lacerated blood vessels or bowel may be necessary. The patient must be informed of potential complications despite the minimally invasive nature of this procedure and should be willing to undergo additional procedures in case of intra-operative complications.
Patients are motivated and, in the case of inflatable prosthesis placement, must possess dexterity to utilize the device effectively. Patients should be properly evaluated before surgery. Patients must have realistic expectations and understand the risks and benefit limitations of prosthetic surgery. Lack of sterile technique, poor placement with crossover of the penile cylinders or improper sizing cause penile deformity, curvature and/or erosion of the prosthesis through the penis or urethra. Penile prosthetic devices may cause postoperative infection even under the best aseptic conditions.
Priapism may result from systemic disease, such as sickle cell anemia, malignancy or pharmacological therapy for impotence. Careful dose titration of the intra- corporeal agent to be administered is essential. Patient selection and education regarding the frequency of dosage, injection technique and complications are essential prior to initiation of self- injection therapy. Aggressive therapy with oral a-adrenergic or intra- cavernosal irrigation is indicated. Patients should be informed of risks and the potential sequela of priapism prior to initiation of intracavernosal injection therapy.
INSTRUMENTATION, WATER AND AIR FILTRATION AND CULTURE MEDIA
Ultrapure water
Distilled and bottled water leach new organics as little as 1 hour after storage. Unfortunately, storage problems are just one of the drawbacks to using distilled or bottled water. There are also problems of higher operating and maintenance costs and contaminant carryover. Laboratory water purification has undergone dramatic changes since the 1980s. Chemists, life scientists and medical technicians are now routinely concerned with impurity levels
that were impossible to measure until recently. Today, deionization, reverse osmosis, carbon absorption and membrane microfiltration are in some respects superior to distillation. Various systems of water ultrafiltration are now commercially available. These systems cost less than distilled or bottled water, and there is no contaminant carryover. Unlike stills, which require regular acid cleaning, ultrafiltration cartridges can be replaced in minutes.
Air filtration
Laminar airflow is airflow in which the entire body of air within a designated space moves with uniform velocity in a single direction along parallel flow lines. Laminar-flow biological safety cabinets (hoods) are devices designed to minimize biohazards inherent in work with low- and moderate-risk biological agents. Vertical laminar-flow containment hoods not only provide product protection by using a filtered downflow of air, but also provide operator protection through a negative-pressure air barrier created along the front opening. With proper aseptic conditions and good laboratory procedures, risk levels are substantially reduced.
Microscopy
Several commercially available microscopes are easy to operate; these include inverted microscopes with a single-laser beam delivered through the objective for gamete micromanipulation, and optional three beam for sperm trapping, tail cutting and/or zona pellucida cutting.