ADVANCES IN FERTILITY TECHNOLOGY
FOR THE
PHYSICAL MEDICINE AND REHABILITATION PATIENT

INTRODUCTION

Spinal cord injured (SCI) patients represent the largest physical medicine and rehabilitation population seen with fertility-related disorders. Eighty percent of these patients are male and have an average age of 30 years at the time of injury. Since approximately 30% of SCI patients are already married at the time of injury, many have not had the opportunity to start a family. Although a small percentage (3%-20%) of patients with spinal cord injuries maintain the capacity to ejaculate, this capacity tends to be unpredictable and is rarely effective in the initiation of a pregnancy. Between 85 to 97% of these men experience permanent loss of ejaculatory function.¹¹ However, many other causes of neurologic injury such as multiple sclerosis, peripheral neuropathies such as diabetes mellitus, and retroperitoneal surgical damage to sympathetic nerves may result in similar anejaculatory states with resultant infertility.

Recent success with inducing ejaculation by means of rectal probe electrostimulation or vibratory induction combined with assisted reproductive techniques have provided these affected couples an attainable goal of producing their own biologic offspring. Unfortunately, the often inherently poor quality of the sperm generated has contributed in the past to poor fertilization and pregnancy rates. However, with the recent introduction of intracytoplasmic sperm injection (ICSI), which involves the direct injection of a single mature sperm into an oocyte, the problems associated with poor sperm penetrating abilities appear to be overcome. The goal of this chapter is to familiarize the reader with the techniques of sperm retrieval and sperm use in the spinal cord injury patient.

SPERM PROCUREMENT TECHNIQUES

PHYSIOLOGY OF EMISSION AND EJACULATION

Ejaculation is a complex, neurologically mediated event distinct from erections consisting of an integrated three-phase process composed of seminal emission, bladder neck closure, and antegrade ejaculation. In the normal state, sensory afferent input from the penis travels from the pudendal nerve to the spinal cord, and is integrated in the cerebral cortex. The efferent response for the ejaculatory reflex involves the thoracolumbar spinal ganglia (T12-L2) forming the hypogastric nerve, which branches into the preganglionic fibers at the level of the prostate, seminal vesicles, and prostate. (See figure1) While sympathetic stimulation results in emission of semen into the urethra and closure of the bladder neck, parasympathetic stimulation causes the antegrade propulsion of seminal fluid.

ELECTROEJACULATION

Anejaculation resulting from damage to the afferent or efferent neural pathways responsible for emission and/or ejaculation can be treated with electroejaculation. In rectal probe electroejaculation, seminal emission depends on the sinusoidal electrical stimulation of sympathetic efferent fibers and smooth muscle. The best site for inducing seminal emission is in front of the bifurcation of the aorta and between the rectum and obturator nerves, where both pre-ganglionic and post-ganglionic sympathetic fibers reside.(Fig. 2) Unfortunately, because electroejaculation does not stimulate the somatically mediated events of ejaculation or coordinate bladder neck closure essential to antegrade emission, pulsatile expulsion of seminal fluid does not occur. Rather, semen either dribbles from the meatus or is deposited retrograde into the bladder.

DEVICES AND PROBES

Over the past 5-10 years, reliable equipment and medically safe methodology utilizing wave-form generators have been developed that deliver oscillating current of either sine-wave or square-wave pulses. Many different types of equipment have been used to deliver electrical bursts or continuous stimuli. Current rather than voltage appears to be the critical factor causing electroejaculation.

Electroejaculation involves the uses of electrodes that are introduced by one of two basic techniquess: (1) electrodes are placed over a gloved finger and then inserted into the rectum, or (2) a rectal probe is used with either longitudinal or circular electrodes of alternating polarity embedded within a cylinder of polyvinyl chloride. With this apparatus, the electrode's surfaces make contact with the rectal mucosa. The smaller, finger-hand electrode is more effective if moved back and forth over the pelvic urethra, ampulla and seminal vesicles during the electroejaculative process. The more commonly used larger rectal probe stimulates all of these areas at once. A probe that has three ventrally located electrodes was developed by Ball in 1976 for use with rams and bulls and was the prototype refined by veterinarian Stephen Seager for use in humans.³ The rectal probes developed by Seager are available in various diameters and have three ventral electrodes for simultaneous stimulation of both vasal ampullae and seminal vesicles. The probes have a built-in thermistor for recording the temperature of the rectal mucosa during electroejaculation to prevent rectal burns resulting from unmonitored overheating.

TECHNIQUE

Patient preparation prior to induction of electroejaculation requires a careful history and physical examination with special emphasis on previous surgery and infections involving the genitourinary system. It is important for planning appropriate anesthesia to determine the level of the lesion and the degree of perineal sensation.

If the urine is sterile, the patients are prophylactically given ciprofloxacin, 500 mg, on the evening prior to their procedure and for 48 hours following the procedure. If a positive culture is found, culture-specific antibiotics are prescribed for one week prior to electroejaculation. Because the acidic pH of urine is very toxic to spermatozoa, sodium bicarbonate, 600 mg, is given orally, beginning the day before the procedure. Those patients who have had a poor response to either vibratory ejaculation or electroejaculation may be given Sudafed, 60 mg orally four times a day, beginning 2 weeks before the procedure, in an effort to augment emission and ejaculation through stimulation of adrenergic innervation. All patients are instructed to use their routine bowel preparation the evening or morning before the procedure (Table 1).

Table 1

The female partner who will be inseminated with the retrieved sperm must be monitored for ovulation using basal body temperature charts, determination of luteinizing hormone (LH) surge with special LH-monoclonal antibody urinalysis kits, or serial transvaginal ultrasounds. The spouse must also be questioned about menstrual periods, ovulation, and possible tubal blockage. The involvement of a reproductive endocrinologist is essential, not only to recommend necessary testing, but also in the possible induction of ovulation using clomiphene citrate or menotropins (Pergonal, Metrodin), and human chorionic gonadotropin (hCG).

The patient for EEJ is positioned in either the lateral decubitus or lithotomy position. General anesthesia is used in those patients with normal sensation or in those with incomplete lesions who are unable to tolerate electroejaculation. Approximately 25% of patients require an anesthetic, and in these patients, care should be taken to use only narcotics and nitrous oxide to avoid agents with sympatholytic activity.

Initially, all patients are catheterized (without lubricant, as this can potentially affect sperm motility) and the bladder washed with a buffered phosphate solution (100 ml Modified Human Tubular Fluid medium containing HEPES buffer, penicillin G, streptomycin sulfate plus 24 ml of Modified Sperm Wash [5% albumin] solution). Approximately 30 ml of washed solution is left in the bladder, and the catheter is removed. Although vibratory stimulation is most effective in patients with upper cord lesions, all patients are given an initial trial of vibration because the lesion may be incomplete.

If vibratory stimulation is not effective, we perform electroejaculation using the Model 12 electroejaculator (G&S Instrument Company, Duncanville, TX) which contains a continuous probe temperature monitor. A preliminary digital rectal examination and anoscopy are performed to rule out any preexisting pathology, and the sphincter is dilated briefly prior to insertion of the probe.

Once the probe has been placed in the rectal vault with the electrodes oriented anteriorly, horizontal upward pressure is used to ensure probe contact with the anterior rectal wall. Stimulations are initiated using the voltage control knob, with an increasing series of voltages using two stimulations for each voltage, and each stimulation lasting approximately 2-4 seconds. Once a maximum voltage of 5 V has been attained, then with each increase, a minimum of 5 V is used as the baseline voltage. If the patient has not ejaculated at a maximum of 10 V, then we use three stimulations at each voltage before proceeding to the next higher voltage. Most patients will ejaculate at less than 20 V and less than 50 stimulations; however, up to 30 V occasionally has been required. Most patients exhibit a small amount of dribbling antegrade ejaculate when electroejaculation is successful, but this is unpredictable, and catheterization is always done to collect any retrograde semen. Because significantly impaired sperm motility and viability are commonly noted in the retrograde ejaculate, efforts should therefore be directed to maximizing the antegrade portion of the electro-ejaculate and optimizing the technique of preserving functional sperm in the intravesical compartment.¹³ The specimens are then immediately transferred to a warming tray. After the probe is removed, anoscopy is again performed to confirm that no injury has occurred.

Adverse effects of the procedure are uncommon and usually consist only of a transient increase in extremity spasms. Rectal injury has been reported, but we have seen no significant injuries in over 180 procedures using the previously described technique. Although autonomic dysreflexia and its attendant consequences are potentially serious complications, the use of nifedipine in patients at risk has significantly diminished these problems.³¹

Most often, semen specimens are washed and subsequently used for intrauterine insemination (IUI) or in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). If large numbers of sperm are present, a portion of the specimen may also be used for endocervical or cervical cap insemination. Excessive debris or blood in the ejaculate usually will be removed by sperm washing or Percoll column separation. Occasionally, if sperm motility is adequate, a sperm swim-up procedure can be employed, and sperm of excellent quality can be recovered. Occasionally, we have cryopreserved sperm from electroejaculation, but poor sperm motility often limits the success of this procedure.

UTILIZATION OF SEMEN AND RESULTS

The results of electroejaculation in terms of sperm harvesting have been excellent, with recent data suggesting that an ejaculate of sufficient quality to use in intrauterine insemination or in vitro fertilization can be obtained in approximately 80% of individuals.²⁹ Most patients tend to have good sperm concentrations, with averages of 180-300 million sperm recovered per ejaculate.(see table 2) The major problem has been that sperm motility has averaged only 11-22% with poor functional characteristics, as well.^6,9

Table 2

Scott Department of Urology, Baylor College of Medicine. Results from 1988-1994.

The poor sperm quality that has been a consistent finding in patients with spinal cord injury is likely due to a number of factors: chronic urinary tract infections and epididymitis as a result of residual urine and infrequent catheterizations, testicular hyperthermia as a result of sitting in the wheelchair and the loss of vasculogenic tone, and infrequent ejaculations with resultant stasis of sperm and prostatic fluid, possible antisperm antibodies, and chronic long-term use of various medications. Repeating ejaculation procedures on successive days may improve sperm motility, but we have not found this to be a predictable occurrence. In addition, it has been suggested that the electroejaculation procedure itself may be detrimental to sperm because of the thermal and electrical effects of the procedure.²⁵ However, recent in vitro studies utilizing a simulated rectal model have failed to confirm this suspicion.³⁵ Several studies have also suggested that up to 50% of patients with spinal cord injury have histologic abnormalities on testicular biopsy.^24,32

The level of the spinal cord injury and the type of bladder management clearly affect the quality of the recovered sperm. Two recent studies reported that men performing intermittent catheterization, in contrast to all other forms of bladder manangement, had higher total sperm counts and motility.^23,27 The reason for this beneficial impact is likely related to the maintenance of a lower pressure bladder with resultant diminished risks of serious infections and reflux of urine into the ejaculatory ducts. Ohl et al also demonstrated that thoracic paraplegics and those with complete injuries had better overall semen parameters.²³

To initiate a pregnancy following electroejaculation, IUI or IVF is required. Although the lower limits of acceptability vary, a minimum of 1 million total motile sperm is usually required for IUI. With other forms of assisted reproductive techniques, conception can occur with a much lower sperm density, as few as 50,000 sperm per oocyte for IVF, and 1 sperm per oocyte with ICSI. Although results with these techniques have been encouraging, an accurate assessment of pregnancy rates has been difficult because of the lack of large published series . The first live birth with electroejaculation and IUI was reported in 1978,¹² and a review of the literature documents 23 total births,^7,20 although the true number is undoubtedly much higher.

Between 1986 and 1994, 152 electroejaculation treatments (107 spinal cord injury, 25 retroperitoneal lymph node dissection, 25 other) were performed at Baylor College of Medicine. An antegrade or retrograde sample was successfully obtained in over 75% of spinal cord injury patients and in 66% of patients with retroperitoneal lymph node dissection. Sixteen (11%) pregnancies resulted with IUI for all patients, and 11 of these pregnancies were in the spinal cord group (10%).

VIBRATORY STIMULATION

Vibratory stimulation, which appears to initiate an ejaculation reflex and usually produces an antegrade ejaculation, is usually effective only in patients with upper cord lesions. However, we suggest that all patients have an initial trial because the lesion may be incomplete. When vibratory stimulation is unsuccessful, electroejaculation often works quite well.⁵

After standard bladder preparation, a hand-held, electrically driven vibrator (Acuvibe Model 6001) is applied initially to the dorsum of the glans and then to the frenulum and penoscrotal area. This slow movement of the vibrator often identifies a "trigger" point, which is often be reproducible from one cycle to another. The selection of a frequency of 100 Hz and a peak-to-peak amplitude of 2.5 mm may be important for successful ejaculation.³⁰ Patients who ejaculate using this form of stimulation usually do so within 5-10 minutes, and these patients will usually exhibit tumescence and pelvic floor contractions prior to ejaculation. In the absence of contractions, we stimulate only for 2-3 minutes before proceeding with electroejaculation. Most patients who respond to vibratory stimulation will exhibit antegrade ejaculation, but catheterization must be performed because these patients frequently have incomplete closure of the bladder neck and may also have a significant retrograde component.

Brindley et al published one of the largest series of penile vibratory stimulation patients and demonstrated ejaculation in 48 of 81 men (59%) with spinal cord injuries of more than 6 months duration (mostly complete).⁵ Poor prognostic indicators included spinal cord injury within the last 6 months²⁸ and the absence of reflex hip flexion. Eleven pregnancies with nine healthy children resulted. Oates et al. observed similar findings, but only in those men with injuries above the T12 level.²²

More recently, Löchner-Ernst from Germany reported on a series of 206 male spinal cord patients, of whom 84% had supranuclear lesions.¹⁷ Semen was obtained by "vibrostimulation" in 65% of all patients. One hundred and two couples desired to initiate a pregnancy with the following results: 62 pregnancies resulted in 40 couples (15 multiple), and 49 healthy children (4 twins) were born. Twenty-one couples collected and inseminated at home by vibrostimulation, 19 in a clinic (6 IVF, 2 ICSI). Most of these patients required only vibratory stimulation, but some had electroejaculation. The authors concluded that vibrostimulation is in most cases effective for semen collection and that the semen quality is better than after rectal electrostimulation. Using advanced reproductive techniques, a substantial number of these patients can initiate a pregnancy (60%).

FERTILITY ISSUES IN FEMALE REHABILITATION PATIENTS

Relatively little has been published regarding fertility in female rehabilitation patients, perhaps because the preponderance of patients with spinal cord injury are men. However, diseases such as multiple sclerosis and rheumatoid arthritis can be debilitating and predominantly affect women of reproductive age. An excellent contemporary review of these problems was published by Monga.¹⁹ The critical issues relate to the rapid and significant physiologic changes attributable to pregnancy and their effects on the underlying disease process. Concerns about medication effects on embryogenesis and difficulties specific to labor and delivery also become important. These issues in disabled women may be quite complex and clearly demand a multi-disciplinary management approach.

The initiation of fertilization and pregnancy, as well as spontaneous abortion rates, do not appear to be significantly altered in women with spinal cord injury.²¹ Significant problems encountered during pregnancy include asymptomatic urinary tract infections and pyelonephritis,^2,14 formation of decubitus ulcers, predisposition to thromboembolic disease, exacerbation of anemia, development of hypotension related to an increased fall in systemic vascular resistance,⁸ pulmonary compromise, and the development of autonomic hyperreflexia. The most potentially life-threatening of these complications is autonomic hyperreflexia, which may occur in 60-85% of women with lesions above T7.^2,14 While preterm labor and a shorter duration of labor do not appear to be increased, autonomic hyperreflexia may be a problem with delivery.³⁴

Multiple sclerosis (MS) does not appear to be exacerbated by pregnancy during the antepartum period, but may worsen during or after delivery. No evidence exists suggesting that MS adversely affects the outcome of pregnancy. In contrast, 75% of women with rheumatoid arthritis will have an improvement during pregnancy, and experience an exacerbation after delivery.¹⁸ Perhaps the most significant concern is risk posed by the use of medications such as aspirin, indomethacin, and prednisone during labor and fetal development.

INTRACYTOPLASMIC SPERM INJECTION

Despite the success of sperm recovery with electroejaculation and vibratory stimulation, the poor quality of the sperm as manifest by poor motility and impaired penetrating ability has limited the results achievable with IUI and IVF. The recent introduction of intracytoplasmic sperm injection (ICSI), which involves the injection of a single sperm into a single oocyte, has revolutionized the practice of male infertility and has distinct applications for the spinal cord injured male. Although the first success using this technique was reported as recently as 1992, ICSI is now being performed by most major IVF centers in the United States.

BACKGROUND

The origins of gamete micromanipulation in general and ICSI in particular can be traced to the late 1970's when initial attempts at IVF culminated in the first successful IVF "test tube baby" in 1979. In IVF, human oocytes harvested from hyperstimulated ovaries are incubated in a culture dish with sperm. Those successfully fertilized oocytes are termed embryos and are transferred into the uterus. Couples with severe male factor infertility were frequently not candidates for IVF or had poor results because approximately 50,000 motile sperm are required per oocyte. Further limitations of IVF for male factor infertility became apparent when up to 500,000 motile sperm per oocyte were required if the patient had poor quality sperm, as in samples obtained through electroejaculation.

Those couples requiring IVF typically have failed at least several cycles of IUI or have severely impaired sperm density and motility (oligoasthenospermia). The first successful application of IVF with electroejaculation was reported in 1988 by Ayers et al.¹ While subsequent reports confirmed the feasibility of IVF with electroejaculation, the poor sperm characteristics often precluded successful oocyte penetration and fertilization.^15,26,33

Interest in the initial types of micromanipulation procedures, such as zona drilling and partial zona dissection (PZD), evolved because of the disappointing results of IVF for the male factor patient. In these procedures, a physical opening is created in the zona pellucida by using chemical "drilling" with acidic Tyrode's solution or by making a microscopic mechanical incision. Because PZD demonstrated the frequent appearance of polyspermy, a lethal condition involving the entrance of more than 1 sperm into the egg, subzonal insertion of sperm (SUZI) gained popularity. However, SUZI, necessitating the microinjection of 5-20 spermatozoa into the perivitelline space (between the zona pellucida and the plasma membrane), still had high rates of polyspermy. The difficulty with polyspermy was finally overcome with ICSI. Because of higher clinical pregnancy rates and broader applicability for severe male factor infertility, ICSI has replaced PZD and SUZI as the micromanipulation technique of choice.

PROCEDURE

ICSI begins with oocyte retrieval using transvaginal ultrasound-guided puncture at the time of optimal follicular development with appropriate hormonal stimulation. After the retrieved oocytes are scored for maturity and incubated for 3-4 hours, the cells of the cumulus and corona radiata are removed by treatment with a hyaluronidase solution. Those oocytes which have extruded the first polar body (indicating the metaphase II state) and are morphologically intact are candidates for ICSI.

Sperm for ICSI in general are typically prepared using a sperm wash, swim-up procedure, or Percoll density gradient centrifugation, depending on the source of the specimen and the sperm characteristics. Sperm may be obtained from the spinal cord injured patient from fresh electroejaculates, frozen electroejaculates, microepididymal or vasal sperm aspirates,⁴ and even testis biopsy specimens. Fertilization rates using frozen and fresh sperm appear to be comparable for ejaculated specimens, but have not been studied in depth for electroejaculated specimens.¹⁰ However, favorable semen cryopreservation (greater than 33% of pre-freeze motility noted after thaw) was present in 5 of 16 electroejaculation cases (31%) in a series by Buch et al.⁶

Micromanipulation procedures are performed using an inverted phase-contrast microscope at 400X. The pipette manipulation is controlled in three-dimensions using hydraulic micromanipulators, either motor or manually controlled. The injection pipettes have outer and inner diameters of approximately 7 µm and 5 µm, respectively, with a beveled tip having an angle of 45o to 50o.

With the sperm and oocytes in the Petri dish, a single motile sperm with grossly normal morphology is first immobilized and then aspirated tail-first into the injection pipette. Proper oocyte positioning involves placing the polar body at the 6 or 12 o'clock position so that the meiotic plate can be avoided when the sperm are injected at the 3 o'clock position. The sperm in the injection pipette is positioned at its very tip prior to injection. The micropipette is pushed through the zona pellucida until the ooplasm is entered.(Figure 3a-d) Approximately 1-2 pl of medium is introduced into the oocyte with the spermatozoa. The needle is then withdrawn.

After 16 to 18 hours of incubation, the oocytes are examined for the presence of fertilization. The identification of 2 pronuclei (2PN) and or extrusion of the second polar body indicate normal 2PN fertilization. Embryo transfer can be performed from 1 to 3 days after oocyte harvest. Depending on maternal age and the reproductive endocrinologist's preferences, generally from 3 to 6 of the morphologically best embryos are transferred to the uterus.

The costs of micromanipulation with ICSI in a standard IVF cycle vary significantly based on institutional practices, but are approximately $10,000-$14,000 per cycle (December 1996). Most of the cost is related to the IVF portion of the procedure.

RESULTS

Our experience between January 1994 and November 1995 with twelve cycles using sperm obtained via electoejaculation in 10 patients (112 oocytes injected) demonstrated a fertilization rate of 49.1%, with a pregnancy rate of 45.5%, in couples who had failed previous IUI. Although the numbers are small, this series is the largest, thus far, for electroejaculation and ICSI. This experience, combined with the overall success of ICSI for severe male factor infertility problems, implies a significant role for ICSI in those spinal cord patients who have failed previous IUI cycles.

With even the most severe of male factor sperm defects treatable using ICSI, female factors such as age and oocyte quality are being examined more closely. While the severity of semen abnormalities demonstrate little correlation with ICSI results, egg number and egg quality are now the main determinants of success. Poorer results with ICSI have been observed in women >40 years. Despite concerns with the possibility of an increased risk of birth defects from ICSI, no convincing data has been presented as compared with a control population.¹⁶ Concerns regarding sex predilection have not become evident.

SUMMARY

Fertility related concerns are significant for the spinal cord injured patient because of his or her young age and loss of the male's ejaculatory function. Fortunately, using electroejaculation and vibratory stimulation, the successful recovery of sperm has become routine. However, because of the poor quality of the sperm obtained, the assisted reproductive techniques of intrauterine insemination and in vitro fertilization have had only modest pregnancy results. The revolutionary technique of intracytoplasmic sperm injection (ICSI), which involves the injection of a single sperm into an oocyte, is particularly applicable for these patients and has been very successful in initiating pregnancies. The potential for improvement in semen recovery and processing is great and must be considered a challenge for the immediate future. Finally, fertility issues in disabled women may be quite complex and clearly need a multi-disciplinary management approach

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