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Postchemotherapy Ejaculatory Azoospermia: Fatherhood With Sperm From Testis Tissue With Intracytoplasmic Sperm Injection

From the Department of Urology, Boston University School of Medicine, Boston, MA; Departments of Urology and of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, CA; and Reproductive Science Center of Boston, Waltham, MA.

Address reprint requests to Robert Oates, MD, 720 Harrison Ave, DOB Ste 606, Boston, MA 02118-2334; email: robert.oates{at}bmc.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To define the success of testis sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) in azoospermic men with a history of chemotherapy.

PATIENTS AND METHODS: In a retrospective study, 23 men with ejaculatory azoospermia and a history of chemotherapy underwent TESE in a search for usable spermatozoa. In six patients cryopreserved tissue and in nine patients fresh tissue provided sperm for an ICSI cycle. Histologic analysis of the testis was performed in all patients. The presence or absence of sperm, fertilization rates with ICSI, and final outcomes of pregnancy were recorded.

RESULTS: Spermatozoa were found on TESE in 15 (65.2%) of 23 men. On histopathology, the predominant pattern observed was Sertoli cell only (47.8%), followed by hypospermatogenesis (30.4%), mixed (17.4%), and late maturation arrest (4.3%). The fertilization rate was 65.2%, and ongoing/delivered pregnancies occurred in 30.8% of cycles. Six healthy boys and four healthy girls have been born to date.

CONCLUSION: Men who are azoospermic and have had prior cytotoxic therapy make up a small subgroup of males with nonobstructive azoospermia. It is important to define and characterize this subgroup and better define their true fertility potential. Approximately two thirds of these men have retrievable testis sperm, which may be used with ICSI to have healthy offspring. This exciting avenue for paternity has heretofore not been available to such patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CURE RATES OF MANY cancers affecting children and young adults have improved significantly with multidrug chemotherapy. For example, men with testicular germ cell tumors enjoy a posttreatment survival of greater than 90%.¹ In addition, other nonmalignant conditions, such as nephrotic syndrome and systemic lupus erythematosus, are also successfully treated with antineoplastic drugs. Predictably, at least 90% of postpubertal young men become acutely azoospermic during cytotoxic therapy. Depending on the type and dose of agents used for treatment, resolution of the azoospermia occurs in approximately 50% of these men over the ensuing 6 months to 5 years.^2-4 For the remainder, severe oligospermia or azoospermia may be a permanent residual effect of treatment.^5-7 Because many such patients survive well into their reproductive years, parenthood is a possible but unattainable goal if sperm was not banked before the induction of therapy.⁸ Even though it is offered, approximately two thirds of testicular carcinoma patients elect not to cryopreserve sperm before therapy.⁹ For the infant or young child who is cured with antineoplastic agents, there is no opportunity for sperm cryopreservation. As a result, in many postchemotherapy patients who are permanently azoospermic, a lack of banked sperm leaves them with only donor insemination or adoption as reproductive options.

Nonobstructive azoospermia (NOA) is a condition reflecting severe spermatogenic compromise such that no spermatozoa are identified in the ejaculate. It is now clear that a certain level of spermatogenesis must be exceeded before spermatozoa are found in the ejaculate. Thus, there are men with sperm production that decreases below this level in whom no sperm are observed in the semen but in whom sperm are found in miniscule numbers in collected testis tissue.¹⁰ During testis sperm extraction (TESE), testicular parenchyma is removed, processed, and microscopically examined for spermatozoa. These individual sperm cells may be used for intracytoplasmic sperm injection (ICSI), an in vitro reproductive technique in which single sperm are mechanically injected into the cytoplasm of single oocytes, with subsequent embryo development and transfer to the uterus.^11-14 TESE will recover spermatozoa in approximately 56% to 87% of men with NOA.^15-18 Currently, sperm recovery rates from specific subpopulations of NOA men with this diagnosis are not well defined. What are the rates of sperm recovery for men with Klinefelter’s syndrome, those postchemotherapy, and those with Y-chromosomal microdeletions? Is it possible to obtain sperm from NOA males who are postchemotherapy? The objectives of this investigation were manifold and novel. First, we characterized in a dual-institutional study the true fertility potential of a well-defined subgroup of NOA men with a history of cytotoxic therapy. We also evaluated biopsy histology and simultaneously performed TESE and calculated the likelihood of recovery of spermatozoa in these men. By use of ICSI with retrieved testicular spermatozoa, the rates of fertilization and ongoing pregnancies were determined. We briefly review the genetic implications of using these sperm with assisted reproduction and the potential risks for the future offspring regarding potential malignancy or congenital malformation. These results should allow the clinician, both oncologist and urologist, to properly counsel postchemotherapy patients with NOA on the likelihood of sperm retrieval and pregnancy with TESE. It informs us, for the first time, that a terrible side effect of curative therapy—the elimination of paternity—may be overcome with a combination of advanced reproductive therapies. This provides immediate hope for men of reproductive age who are presently in this predicament and future hope for those who are currently undergoing spermatotoxic treatment. This in no way, however, eliminates the need to offer sperm banking to all postpubertal males before the induction of treatment. Electroejaculation is a possible alternative for semen procurement if masturbation is not successful.¹⁹

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study consisted of patients with postchemotherapy NOA (Table 1) from two institutions—Boston University Medical Center and the University of California San Francisco (UCSF)—for whom there was no cryopreserved sperm available for use. Institutional review board approval was obtained at both institutions for this study. A group of 23 men (mean age, 35.8 years; range, 25 to 50 years) was identified as the appropriate population. All men presented with normal volume azoospermia, confirmed by semen pellet analysis (microscopic examination of the pellet after centrifugation of the semen sample). A comprehensive history was taken and physical examination performed on all patients. We specifically tried to identify the reason for cytotoxic treatment, age at therapy (range, 1 to 39 years), and specific agents given. None of the 23 patients had pretreatment semen cryopreservation. All patients had normal sexual libido and erectile function. Physical examination demonstrated small testicles unilaterally (testis cancer patients) or bilaterally with an abnormally soft consistency in all men. The vasa deferentia and epididymides were palpably normal, with no evidence of obstruction. Laboratory plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, and testosterone were evaluated in most patients.

Boston Medical Center: TESE, Preparation and Cryopreservation of Testicular Homogenates, and ICSI
Ten patients underwent TESE, wet preparation, and diagnostic testicular biopsy for histopathology. After sterile preparation and draping, local anesthetic block of the spermatic cords and scrotal incision site was performed with 30 mL of 0.25% bupivacaine. An ophthalmic knife was then used to create a 1- to 2-cm transverse defect within the tunica albuginea near the upper pole of the testicle, carefully avoiding the vascular supply. With gentle pressure, extruding seminiferous tubules were excised and placed within a few drops of test yolk buffer (TYB; Irvine Scientific, Santa Ana, CA) on a sterile glass slide. After mincing of the testicular tissue on the glass slide, this specimen was then transferred into a sterile test tube containing TYB and labeled appropriately before transfer to the embryology laboratory for examination, possible sperm extraction, and potential cryopreservation. The remaining fluid on the slide was then evaluated under a microscope with x400 magnification as the wet preparation. If no sperm were identified, the same procedure was performed on the contralateral testicle. Before closure of the tunica albuginea, a separate small specimen of testicular tissue was removed and sent for histopathology.

On arrival at the laboratory, the biopsy specimens were allowed to settle. A 5-µL aliquot of the supernatant was microscopically examined for the presence of spermatozoa. If spermatozoa were found, multiplication by 200 provided an estimate of the concentration of spermatozoa per milliliter. Glycerol (Sigma, St. Louis, MO) was added to the supernatant to adjust the final concentration to 10% glycerol (vol/vol). The biopsy material was then transferred to a sterile 5-mL polystyrene test tube together with 1.0 mL of TYB/glycerol 10% (vol/vol). The tissue was grossly dissected with sterile iris scissors. The minced tissue was then homogenized by repeated aspiration through 16- and 18-gauge hypodermic needles. A 5-µL sample was searched by using phase contrast microscopy for spermatozoa, and the number per milliliter was calculated as described previously. If spermatozoa were seen, the homogenate was adjusted to a final volume of 5.0 mL with TYB/glycerol 10% (vol/vol), and 500-µL aliquots were transferred to sterile, labeled polypropylene tubes (Nunc Cryovials; USA Scientific Plastics, Ocala, FL). Prepared homogenate and supernatant vials were then cooled to 4°C in a refrigerator. By use of a programmable freezer, all vials were cooled at -1°C/min to -30°C and finally to -150°C at -5°C/min. Vials were then transferred directly to liquid nitrogen and stored pending isolation of oocytes from the female partner.

If sperm retrieval was successful, the female partner subsequently underwent standard ovulation induction. On the day of oocyte isolation, a single vial of cryopreserved tissue was thawed, and individual sperm were isolated, immobilized, and used for injection. Only spermatozoa that showed twitching or motility or that demonstrated sperm tail swelling under hypoosmotic conditions were selected for use. After documenting fertilization and embryo formation, intrauterine transfer was performed 2 to 5 days later. Careful observation and follow-up were then performed throughout the pregnancy, and the outcomes were recorded. These procedures have been detailed previously.²⁰

UCSF Medical Center: Testis Fine-Needle Aspiration Mapping and TESE, Preparation of Testicular Homogenates, and ICSI
A different strategy is used at UCSF to determine which men with failing testes are candidates for ICSI. At UCSF a program of systematic, diagnostic fine-needle aspiration (FNA) sampling of the testis without a formal biopsy is undertaken in the office before TESE.^21,22 Information obtained from the FNA "map" is then used to guide subsequent formal TESE procedures performed at the time of egg retrieval and ICSI.²³ Briefly, the scrotal skin and spermatic cords were locally anesthetized and the scrotal skin stretched taut over the testis. Planned aspiration sites were marked on the scrotal skin overlying the testis, approximately 5 mm apart. Percutaneous FNA was performed with a 1-inch, 23-gauge needle (Becton Dickinson Co, Franklin Lakes, NJ) by using the established suction-cutting technique.²⁴ A 10-mL syringe (Becton Dickinson) was placed on suction in a Cameco syringe holder (Precision Dynamics Corp, San Fernando, CA). Precise, gentle, in-and-out movements varying from 5 to 8 mm were used to aspirate tissue, which was then expelled onto a slide, smeared, and fixed in 95% ethyl alcohol. A routine Papanicolaou stain was performed, and each slide was interpreted by an experienced cytopathologist for specimen adequacy and the presence or absence of mature sperm with tails. Those patients in whom sperm were found were advised to proceed with subsequent TESE on the day of oocyte retrieval.

On the basis of the FNA results, the three or four most likely sites for sperm recovery were identified and the TESE biopsies directed to these areas.²³ All TESE procedures were performed under local anesthesia and involved localized, avascular incisions in the testis tunica albuginea. However, after mapping, the tunica albuginea harbors small (< 1 mm) raised scars, almost pocklike, that corresponded to FNA puncture sites. These are easily identified and form a grid that guides the TESE incision to sperm-rich areas. Single, relatively small biopsies were taken from each preplanned site, and the tissue was immersed into 1.0 mL of Earle’s medium (Gibco BRL, Grand Island, NY) supplemented with 4 mmol/L of sodium bicarbonate, 21 mmol/L of HEPES, 0.47 mmol/L of pyruvate, and 10% vol/vol synthetic serum substitute (Irving Scientific, Santa Ana, CA) and maintained at 37°C in a sterile, 2.5-cm petri dish (Falcon 3001; Becton Dickinson, Lincoln Park, NJ). The specimen was lightly macerated with sterile scalpel blades, transferred to the embryology laboratory, and examined with inverted microscopy for sperm.

Morphologically normal and motile sperm were recovered with a "swim-out" technique in which 5 µL of undiluted supernatant from the tissue suspension was pipetted to the center of a microdroplet (7 µL) of Earle’s medium. Spermatozoa reaching the microdroplet edge were collected with an finely drawn ICSI pipette (Pacific Andrology Inc, Montrose, CA), transferred to polyvinylpyrrolidone (Irvine Scientific) for tail breaking, and injected directly into oocytes. Excess sperm were cryopreserved in a manner similar to that described earlier. Ovulation induction, sperm microinjection, embryo culture, and embryo transfer were performed in a standard fashion.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
History of Chemotherapy
Of the 23 patients, three (nos. 1 to 3; 13%) had prepubertal chemotherapy, whereas 20 (nos. 4 to 23; 87%) received chemotherapy postpubertally (Table 1). Patient nos. 2 and 3 underwent chemotherapy for nonmalignant conditions. Patient nos. 5 to 8, 10, 14 to 16, 19, and 21 to 23 were given therapy for testis cancer. Patient nos. 1, 4, 9, 11 to 13, 17, 18, and 20 were treated for other neoplastic conditions. As noted in Table 1, an analysis of the cytotoxic regimens used in these patients reveals that 18 (78.3%) of 23 patients were treated with alkylating agents (cyclophosphamide, ifosfamide, dacarbazine, chlorambucil, lomustine, mechlorethamine, cisplatin, or procarbazine). The number of cycles and total dose of the particular drugs are listed in Table 1.

Hormonal Variables
The mean serum FSH was 21.7 mIU/mL (range, 5.7 to 60.7 mIU/mL), mean luteinizing hormone was 8 mIU/mL (range, 3.4 to 20.9 mIU/mL), and mean testosterone was 347.9 ng/dL (range, 124 to 511 ng/dL; Table 1).

Histology Results
Testicular histopathology revealed a pattern of Sertoli cell only in 11 (47.8%) of 23 patients (Table 1). Also noted in these cases was a moderate amount of peritubular fibrosis. Only one of these patients revealed Leydig-cell hyperplasia. Seven patients (30.4%) had hypospermatogenesis, and four patients (17.4%) had a mixed pattern with germinal aplasia as the major component, with rare tubules containing spermatogonia, spermatocytes, and an occasional spermatid. One (4.3%) of the 23 patients had late maturation arrest on histology.

TESE/ICSI Results
Sperm was successfully extracted in 15 (65.2%) of 23 patients (Table 2). For patients at Boston Medical Center, six to 10 vials containing 500 to 5,000 sperm per vial were cryopreserved from each patient if sperm were detected. In patient no. 1, bilateral TESE revealed sparse sperm from the right side only and none on the left. A coordinated cycle of TESE and ICSI was performed several months later, and no sperm was found in either the freshly obtained or cryopreserved tissue; this was not an unexpected result. A total of 26 ICSI cycles were performed in these 12 couples: patient nos. 5, 7, 10, 14, 19, and 21 had one cycle; patient nos. 4, 6, and 12 had two cycles; patients 15 and 18 had three cycles; and patient 13 had eight cycles. In patient no. 13’s first ICSI cycle, a twin pregnancy was lost at 25 weeks’ gestation secondary to premature rupture of the membranes, with no gross abnormalities of the fetuses noted. In patient no. 6’s first ICSI cycle, the pregnancy was lost at 6 weeks. Patient no. 19 has an ongoing twin pregnancy. Overall, the fertilization rate with cryopreserved sperm ranged from 25% to 100%, with a mean of 65.2%. The ongoing/delivery rate for these 11 couples is currently 30.8%. All babies born so far have had normal newborn pediatric examinations, with no adverse findings noted.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Almost every chemotherapeutic agent has the untoward side effect of reproductive tissue damage.²⁶ In general, chemotherapeutic agents kill rapidly dividing cells, certainly a characteristic of germinal stem cells (type A and B spermatogonia) and of primary and secondary spermatocytes. This may result in permanent, nonobstructive ejaculatory azoospermia, depending on the agents and dosage used and the fraction of stem cells lost.^27-29 In general, nearly all cancer patients are azoospermic and demonstrate increased FSH values within months of starting treatment. Resumption of spermatogenesis to the degree that spermatozoa are in the ejaculate occurs in approximately 50% of patients after 6 months to 5 years.^30-33 Recovery of spermatogenesis depends on the level of spermatogonial stem-cell kill and the rate at which the surviving stem cells can repopulate the seminiferous tubule and resume differentiation. However, men with testis cancer often have reduced sperm counts at the time of discovery of their cancer. Jacobsen et al³⁴ described 60 men who underwent unilateral orchiectomy and were placed on a surveillance protocol with no interval chemotherapy or radiotherapy. Around the time of their surgery, 36 of the men were normospermic, 17 were oligospermic, and seven were azoospermic. After 1 year, seven (12%) remained azoospermic, demonstrating that a small proportion of men with testis cancer have preexisting and permanent severe spermatogenic deficiency in the remaining gonad. In those who undergo chemotherapy for metastatic disease, it is impossible to determine whether resultant azoospermia is consequent to a baseline spermatogenic problem, chemotherapy, or a combination of both.

Among the various chemotherapeutic agents, procarbazine (patient nos. 11 to 13) and the alkylating agents (patient nos. 1 to 5, 7 to 13, 15, 18 to 20, 22, and 23), such as cyclophosphamide, cisplatin, mechlorethamine, and chlorambucil, tend to cause more serious and permanent spermatogenic dysfunction.^29,35-37 Another important consideration is the total cumulative dosage of the agents received. Meistrich et al³⁸ indicated that if the dosage of cyclophosphamide was less than 7,500 mg/m², approximately 70% of men recovered baseline spermatogenesis, whereas only 10% of those who received higher doses recovered similarly. DeSantis et al⁴ documented that with cisplatin treatment more patients recovered spermatogenesis when the total dosage was less than 400 mg/m². Furthermore, Pont and Albrecht³⁹ noted that irreversible infertility occurs in patients who are given chlorambucil at doses of 400 mg. It was also observed that ifosfamide was less toxic to type A spermatogonia than other alkylating agents and that antimetabolites, topoisomerase inhibitors, bleomycin, vinca alkaloids, and dacarbazine were significantly less cytotoxic than chlorambucil. Somewhat contrary to this, Rautonen et al⁴⁰ observed that even vincristine may have a significant negative effect on long-term gonadal function. Taken together, these and other studies demonstrate that if the number of chemotherapy cycles and total dosages are reduced, spermatogenic recovery is more likely.

It is important to note that all studies in the literature to date have focused on sperm and fertility outcomes with ejaculated spermatozoa. However, ejaculatory azoospermia is not the same as testicular azoospermia. Therefore, studies on the reproductive tract toxicity of various chemotherapeutic agents have limited validity in the era of assisted reproductive technology in which it is possible to use testis sperm to conceive. The level of spermatogenesis necessary for sperm to be present in the testis is likely to be far less than that required for sperm in the ejaculate. Indeed, this study vividly illustrates this difference. It is hoped that further work will define the determinants for testicular azoospermia in terms of drug dose, class of chemotherapeutic agent, duration of therapy, and timing of administration (pre- or postpubertal).

It has been reported that TESE has a sperm recovery rate of approximately 56% to 87% in patients with NOA from various causes.^12-14 It has also been demonstrated that age, increased FSH, small-volume testicles, and histopathology do not predict the likelihood of successful TESE.¹⁸ Our subpopulation of NOA men indicates similar rates of TESE success. Furthermore, 78.3% of the patients were treated with alkylating agents, procarbazine, or both, drugs known to be particularly spermatotoxic. There is scarcely any literature that addresses the paternity potential in these men by using testis sperm. In a case report, Res et al⁴¹ presented a patient who underwent successful TESE/ICSI by using pretreatment-cryopreserved testis sperm. Naysmith et al⁷ reported TESE results from five patients who had chemotherapy-induced NOA. However, fertilization and pregnancy results were not well defined in this cohort. Our overall fertilization rate of 65.2% and ongoing/delivered rate of 30.8% compares favorably with the success achieved in other men with NOA.

Because there are mutational effects on spermatogonial DNA, concerns often arise as to whether the offspring will be affected with cancer or congenital malformations after spermatogenesis recovers. Several large studies have analyzed spontaneous pregnancy outcomes in patients previously treated with chemotherapy and have noted no increase in congenital malformations or other diseases and have noted normal offspring karyotypes.^42-45 Mulvihill et al⁴⁴ demonstrated that the offspring of cancer survivors had a 0.3% chance of developing cancer, and this is similar to controls. However, can we be as confident with the results of spermatozoa from men who have only testicular sperm remaining after cytotoxic drug treatment? Are surviving spermatogonia genetically intact but simply fewer in number after chemotherapy? Are they genetically damaged, yet able to make it through important quality control checkpoints during spermatogenesis? The finding of healthy babies in this study is certainly encouraging in this regard, but further follow-up of these children is important.

In conclusion, our dual-institution study is the first to characterize and determine the fertility potential of postchemotherapy men with NOA. These men do have a chance to achieve biologic fatherhood. We noted that despite the lack of any germ cells on histology, the majority of these patients can have sperm recovered with TESE, regardless of age, reason for treatment, FSH level, and physical examination findings. Future studies will reveal whether there is or is not an increased risk of genetic or congenital malformations among offspring. Future research should address whether spermatid or spermatocyte injections will produce normal offspring, and the possibility of using cryopreserved testicular tissue from prepubertal patients for ICSI after in vitro germ cell culturing or germ cell transplantation.

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Submitted April 25, 2001; accepted September 26, 2001.

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