Abstract
Objective: To confirm the feasibility of using fresh or frozen/thawed spermatozoa from cancer and collagen diseased patients for intracytoplasmic sperm injection (ICSI).
Patients: Two participants were diagnosed with testicular carcinoma and one patient was diagnosed with collagen disease.
Methods: Of the two carcinoma patients, one patient provided a fresh testicular biopsy sample and one patient ejaculated fresh sperm after surgical operation. One collagen diseased patient's sperm was frozen, and three samples were used in subsequent ICSI treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for the retrieval of oocytes.
Results: Two patients with testicular cancer and their respective partners achieved successful pregnancies from ICSI using fresh sperm after surgery. The patient suffering from collagen disease had a successful pregnancy from ICSI using sperm frozen prior to chemotherapy treatment.
Conclusions: Patients with testicular carcinoma or collagen disease who might desire to father children in the future should be offered sperm preservation prior to the initiation of chemotherapy treatment. Under currently available treatment, patients with cases of severe disease can still become biological fathers. (Reprod Med Biol 2004; 3: 69–75)
Keywords: azoospermia, cryopreservation, intracytoplasmic sperm injection, malignant disease
INTRODUCTION
CURRENT MEDICAL TECHNOLOGY has made tremendous advances; however, some patients with severe diseases, such as cancer and collagen disease, still present therapeutic challenges. Testicular cancer is the most frequent malignant tumor in men aged 20–40 years. 1 Non‐seminomas are the most frequent type found in the age group of 10–34 years, whereas seminomas are the predominant testicular tumor found in older men (35–59 years). 2 Even though the majority of these patients are young before cancer treatment is initiated, it is important to consider the consequences of treatment, which may impact the quality of life, including fertility.
Chemotherapy and irradiation therapy damage the germ cells, while surgical treatment impairs the ability to ejaculate; however, Novero et al. achieved successful fertilization of oocytes and a pregnancy after intracytoplasmic injection of spermatozoa extracted from testicular tissue samples of a patient with a seminoma. 3 Van Basten et al. demonstrated contralateral testicular tumors in 3% of 365 patients with non‐seminoma testicular tumors. 4 Furthermore, testicular cancer patients have a very good rate of long‐term survival; almost 100% of patients with local/regional disease are cured, and 70–80% of patients with advanced disease are cured. Therefore, if semen can be frozen prior to treatment, testicular cancer patients will retain the ability to father children.
In the current report, we present one collagen diseased patient whose sperm was obtained from ejaculation prior to chemotherapy treatment. One of the two cancer patients had sperm obtained from ejaculation after surgery, and another patient had sperm obtained from a testicular biopsy after surgical and chemotherapy treatment. These sperm were of two varieties: just thawed or fresh, and used for ICSI. A successful pregnancy outcome was achieved in all three cases. Informed consent was given in each case.
MATERIALS AND METHODS
Retrieval of oocytes
PATIENTS WERE TREATED first with oral contraceptive pills (Planovar, one tablet/day) beginning on the third day of the menstrual cycle until day 23 of the cycle, and from day 17, patients were treated with gonadotropin‐releasing hormone agonist (GnRH‐a; Suprecure, Aventis, Tokyo, Japan) 900 µg/day until human chorionic gonadotrophin was given on day 12 (hCG; hCG‐MOCHIDA, Mochida, Tokyo, Japan). After discontinuance of Planovar on day 23, usually within several days patients began to stop bleeding (this menstrual cycle was a treated cycle), and on the third day of the cycle the human menopausal gonadotropin (hMG; Humegon, Organon, Hague, the Netherlands) was administered 300 IU/day for the duration of approximately 8 days. Ovulation was induced by the administration of 10 000 IU of human chorionic gonadotropin (hCG; Profasi, Serono, Geneva, Switzerland) when the average size of the dominant follicle was 20 mm. Approximately 36 h after hCG administration, oocyte retrieval was performed by vaginal ultrasound‐guided follicular puncture under general anesthesia.
The cumulus corona cells were initially removed through exposure to 60 IU/mL of hyaluronidase for up to 1 min. Only metaphase II (MII) oocytes were injected for ICSI. 5 , 6
For luteal support, the patient inserted a 200 mg progesterone vaginal suppository twice a day for 16 days, beginning 24 h prior to the embryo transfer.
Retrieval of sperm from testicular tissue
Biopsy was taken from an equatorial location and not from regions involving the rate testes. A small piece (5 × 5 mm) of extruding testicular tissue was excised and minced using sterile glass slides in a dish with 1 mL modified‐human tubal fluid (m‐HTF) medium. An inverted microscope (×400) was used to check for motile sperm.
The dissection medium was carefully aspirated to avoid aspirating any tissue and centrifuged for 5 min at 750 g. The pellet was resuspended in 1 mL of medium. The sperm suspension was kept in an incubator until the ICSI procedure, as incubating testicular sperm for several hours is known to improve sperm motility. 7
Cryopreservation of sperm and thawing
The semen were mixed with equal volumes of TEST‐Yolk buffered with glycerol (Irvine Scientific, Irvine, CA, USA), and exposed to liquid nitrogen (LN2) vapor for 15 min before being immersed in LN2 for storage.
When the sperm needed to be thawed, the frozen sample tube was warmed at room temperature for 10 s, and placed directly into a 30°C water bath, where thawing was completed. We then centrifuged it with Percoll. The pellet was resuspended in 0.2 mL of serum containing 10% HTF, and then incubated at 37°C under 5% CO2 in air for 2–3 h. Thereafter, an inverted microscope was used to check for motile sperm that could be used for ICSI. Any motile sperm were removed and transferred to a new medium using an injection pipette.
Cryopreservation of embryo
The vitrification and thawing of embryos was performed according to the vitrification technique that involves ethylene glycol (EG), dimethyl sulfoxide (DMSO), Ficoll and sucrose as cryoprotectants for rapid vitrification using a cryoloop. Thus, for vitrification, the embryos were equilibrated in 10% EG plus 10% DMSO for 2 min at room temperature, and then placed into vitrification solution of 20% EG, 20% DMSO and 10 mg/mL Ficoll 70 with 0.65 M sucrose. Finally, the embryos were plunged immediately into LN2 for approximately 20 s. For the thawing procedure, the embryos were immediately placed into 0.5 M sucrose in solution for 2 min, and sequentially placed into 0.25 M for 3 min, and then removed into 0 M sucrose for 5 min at 37°C condition. The thawed embryos were transferred to patients with treatment of hormone replacement. 5
Culture to the blastocyst stage
The sequential media approach was used for the extended culture to the blastocyst stage. The embryos were initially cultured in 20 µL K‐Sydney IVF Cleavage Medium (Cook, Brisbane, Australia) until day 3. On day 3, embryos were transferred for further development into 30 µL drop blastocyst K‐SIBM medium (Cook). On the night prior to transfer, the dishes were prepared and preincubated at 37°C in a 6% CO2, 5% O2, 89% N2 humidified environment.
Cleaving embryos were selected for early transfer based on their morphologic score. If patients requested to continue more culture time, the early stage embryos from the each group were reserved for extended culture. Blastocysts were transferred on day 5, and after transfer, supernumerary blastocysts were frozen by vitrification. 8
Assessment of embryo quality
The embryo quality was evaluated in embryos with equal‐sized blastomeres, an ideal cleavage rate (four cells on day 2 and eight cells on day 3), and percentage of fragmentation were defined as grade 1–5 on early embryo stage according to morphology by biological light microscope with modified Veeck's criteria. 9 The quality of blastocysts morphology was evaluated by Gardner's criteria. 10
RESULTS
Case 1
IN AUGUST 1998, a couple (33‐year‐old husband; 31‐year‐old wife) was referred to our clinic (Ladies Clinic Kyono, Miyagi, Japan). The man weighed 71 kg and was 180 cm tall in height (Table 1). He had been diagnosed with dermatomyositis (collagen disease) and was treated with the following drugs: Predonine, 25 mg; Warfarin, prostaglandine E, Marzulene, Glymor and Cinal. At that time, the patient was diagnosed with the possibility of lung cancer, therefore he requested cryopreservation of his semen. From August to October 1998, his semen was frozen six times. The average semen analysis included: a volume of 4.2 mL; a concentration of 17 × 106/mL; a motility of 62.9% and an abnormal morphology 17.9% (Table 2).
Table 1.
Patients’ aspects for two participants who were diagnosed with testicular carcinoma and one patient who was diagnosed with collagen disease
| Patient | Case 1 | Case 2 | Case 3 |
|---|---|---|---|
| Patient age (years) | 33 | 28 | 35 |
| Weight (kg) | 71 | 64 | 80 |
| Height (cm) | 180 | 172 | 175 |
| Type of diagnosis | Dermatomyositis | Seminoma | Testicular cancer |
| Treatment | Chemotherapy | Extirpation and chemotherapy | Extirpation and chemotherapy |
Table 2.
Sperm characteristics for two participants who were diagnosed with testicular carcinoma and one patient who was diagnosed with collagen disease
| Case number | Average of semen parameter† | Semen parameter when pregnancy ensued | Semen parameter after thawing | Semen parameter after swim up | |
|---|---|---|---|---|---|
| 1 | Volume (mL) | 4.2 | 5.2‡ | 1.0 | 0.5 |
| Count ×106/mL | 17 | 26 | 12 | 2 | |
| Motility (%) | 62.9 | 65.4 | 58.3 | 100 | |
| Abnormal morphology (%) | 17.9 | 7.7 | 0.0 | 0.0 | |
| 2 | Volume (mL) | 4.5 | 5.0 | – | 2.0§ |
| Count ×106/mL | 21.5 | 6.0 | – | 4.0 | |
| Motility (%) | 66.5 | 80.0 | – | 91.3 | |
| Abnormal morphology (%) | 14.8 | 13.3 | – | – | |
| 3 | Volume (mL) | – | – | – | – |
| Count ×106/mL | – | – | – | 10¶ | |
| Motility (%) | – | – | – | 100 | |
| Abnormal morphology (%) | – | – | – | 0 | |
Average was before freezing sperm.
‡Frozon sperm when used for intracytoplasmic sperm injection (ICSI).
§Fresh sperm when used for ICSI.
Testicular sperm; sperm 10/one sight microscope (×400). Case 1, frozen sperm; case 2, fresh sperm; case 3, fresh sperm.
In February 1999, his wife conceived by natural coitus; however, the pregnancy was a hydatidiform mole. We froze his semen on six occasions and performed artificial insemination (AIH; five cycles) from October 1998 to February 2000. A successful pregnancy did not ensue; therefore, we next performed ICSI. In April 2000, we thawed frozen sperm which we had cryopreserved in September 1998, and used for ICSI. The thawed sperm recovered had a concentration of 12 × 106/mL; a motility of 58% and an abnormal morphology 7.7%.
Finally, swim up sperm using ICSI had a concentration of 2 × 106/mL; a motility of 100% and an abnormal morphology 0% (Table 2).
As his wife had a completely normal physical examination, controlled ovarian hyperstimulation was performed using the long protocol.
Of 12 retrieved oocytes, 11 were MII oocytes, and 10 were fertilized, and 10 cleaved embryos developed all grade 1 on day 3, and we removed them into blastocyst‐medium until day 5, and then nine of 10 embryos formed high quality blastocyst; 3AA on day 5. 11 Three of nine 3AA blastocysts were transferred to the wife of case 1 on day 5, and supernumerary embryos were vitrified (Table 3).
Table 3.
Clinical results when pregnancy ensured
| Case 1 (%) | Case 2 | Case 3 (%) | |
|---|---|---|---|
| Number of total oocytes retrieved | 12 | – | 28 |
| Fertilization rate (two pronuclei/metaphase II) | 10/11 (90.9) | – | 18/24 (75) |
| Cleavage rate (cleaved/two pronuclei) | 10/10 (100) | – | 18/18 (100) |
| Blastocyst development rate on day 5 (blastocyst/two pronuclei) | 9/10 (90) | – | 10/18 (55.5) |
| Good blastocyst quality rate† | 9/9 (100) | – | 6/10 (60) |
| Number of blastocyst transferred | 3 | – | 2 |
| Number of frozen blastocyst | 6 | – | 8 |
| Number of gestational sac | 1 | 1 | 1 |
| Number of frozen‐thawed embryo | – | 4 | – |
| Number of survival embryo | – | 4 | – |
| Number of frozen‐thawed embryo transferred | – | 2‡ | – |
Transferred three blastocysts were all ≥3AA by Gardner's criteria. 11
‡Transferred two embryos were all G1 by Veeck's criteria.
Fourteen days after oocytes retrieval the serum hCG level was 695.3 mIU/mL, and 21 days after oocyte retrieval, a fetal heart beat (FHB) within a gestational sac (GS) was present. At a gestational age of 30 weeks and 1 day, she delivered a single male infant with no congenital malformations by cesarean section.
Case 2
In August 1996, a couple (28‐year‐old husband; 26‐year‐old wife) was referred to our clinic; the woman had failed to conceive despite three AIH treatment cycles, conducted at different clinics. The husband weighed 64 kg and was 172 cm tall (Table 1). The couple achieved one pregnancy by natural coitus; however, a spontaneous abortion ensued. In September 1996, we attempted conventional IVF using ejaculated sperm; of 13 retrieved oocytes, nine were fertilized, and five cleaved embryos were grade 1 and the other two embryos were grade 3. Three of the grade 1 embryos were transferred, and supernumerary embryos were cryopreserved; however, a pregnancy did not ensue. Subsequently we thawed frozen embryos and recovered one grade 1, and two grade 3, these three frozen/thawed embryos were transferred; however, again pregnancy did not ensue. In 1997, we discovered a left‐sided testicular tumor and testicular biopsy revealed a seminoma. In January the patient had his left testis removed. Prior to the chemotherapy after operation, we obtained and cryopreserved a semen specimen on January 1997. The average semen analysis included: a volume of 4.5 mL; a concentration of 21.5 × 106/mL; a motility of 66.5% and an abnormal morphology 14.8% (Table 2). This specimen was used on June 1997.
The patient underwent a left orchiectomy and received chemotherapy for 2 months after collection of sperm. In May 1997, immediately after the surgery, an ejaculated specimen was azoospermic. However, approximately 3 months later, in August 1997, spermatogenesis had partially recovered. Semen analysis included: a volume of 3.5 mL; a concentration of 1.3 × 106/mL; and a motility of 84%. This specimen was frozen, however, it was used for ART treatment.
On June 1997, a second ICSI treatment cycle was performed using frozen sperm which was frozen prior to chemotherapy after operation on January 1997; however, none were successful. Of 22 retrieved oocytes, 16 were MII oocytes, and 11 were fertilized, and nine cleaved cells developed; five embryos were grade 1, and the other two embryos were grade 2 and two embryos were grade 3. Three embryos, two grade 1 and one grade 2 were transferred, and supernumerary embryos were frozen; however, a pregnancy did not ensue. In September 1997 we thawed frozen embryos and from these we recovered only one grade 3, and transferred it, however, again pregnancy did not ensue.
In July 2000, the latest oocyte retrieval, of 10 retrieved oocytes, seven were fertilized using ejaculated fresh sperm (Table 2); (volume: 5.0 mL; concentration: 6 × 106 mL; motility: 81% and abnormal morphology: 13%), and four 2 pronuclei (2 PN) stage embryos were frozen, three embryos developed 6–10 cleaved cells on day 3, these three embryos; all grade 1, were transferred on day 3. However, pregnancy did not ensue. In January 2001, we thawed frozen 2 PN embryos and cultured until day 2, two embryos were grade 1 and another two embryos were destroyed (Table 3). Two embryos were transferred to case's 2 wife in a natural cycle. Fifteen days later we confirmed a viable pregnancy with a serum hCG level of 1364.4 mIU/mL. In October 2001, at 41 weeks and 2 days’ gestation, the woman vaginally delivered a male infant with no congenital malformations.
Case 3
In 1996, a couple (35‐year‐old husband; 32‐year‐old wife) had achieved a natural pregnancy, delivered by cesarean section. He weighed 80 kg and was 175 cm tall (Table 1). In June 1999, the husband underwent a right orchiectomy and chemotherapy for a seminoma. The chemotherapy of Cisplatin and Bleomycin, was administed from June 1999 to June 2000 as nine courses of chemotherapy. In December 2000, the patient experienced a recurrence at the location of the original surgery; he underwent a retroperitoneal lymph node resection. Following the surgery, he experienced difficulty with ejaculation. In September 2002, the couple was referred to our clinic for infertility treatment. We suspected that his azoospermia was a result of surgical trauma rather than retrograde ejaculation.
We could not recover sperm from ejaculated semen; therefore, in November 2002, we retrieved testicular sperm via a left testicular biopsy, in which we easily found motile sperm; approximately 10 sperm per one sight on the slide glass in minced testicular tissue under the 400× magnification, this fresh sperm was used for ICSI.
We stimulated his wife's ovaries under the long protocol. Of 28 retrieved oocytes, 24 were MII oocytes, and 18 of the 24 MII oocytes were fertilized by ICSI using fresh testicular sperm obtained as mentioned above on November 2002. All of the fertilized embryos were developed into three to eight cells stage embryos on day 3, all embryos were removed into blastomedium, and 10 of 18 were developed to blastocyst stage; six good quality (more than 3BB) and four blastocysts were less than 3CC. 11 Two excellent quality; 4 bc and 5AB were transferred on day 5, and the supernumerary embryos were vitrified (Table 3). 11
The serum hCG level was 260.6 mIU/mL 15 days after oocyte retrieval; a single FHB was visualized in a 6‐week GS. In July 2003, the woman vaginally delivered a healthy female infant with no congenital malformations at 36 weeks and 5 days’ gestation.
DISCUSSION
SEVERE DISEASES, such as many malignant types of cancer and collagen disease impact spermatogenesis. Many patients who have been treated for the above‐mentioned diseases have long‐term survival after chemotherapy and irradiation therapy, and currently a relatively large number of male children and adolescents would benefit from cryopreservation of their semen prior to cancer treatment. If the patient is an adult, there are less challenges; the use of frozen‐thawed sperm as well as ejaculated sperm from these individuals is well established in IVF programs.
Approximately 90% of children and adolescents diagnosed with testicular cancer are cured, but many have permanent gonadal damage, depending on the type of treatment. 12 , 13 It is extremely important for the quality of life in these survivors to maintain fertility and a normal sex life.
In post‐pubertal boys and men, cryopreservation of semen for ART is clinically well established and widely used for infertile males.
Testicular sperm extraction (TESE) specimens and ejaculated sperm has been applied to cancer patients in clinical practice. 14 In Japan, oral presentations have covered the topic; furthermore, there are only a few published case reports describing the use of frozen‐thawed spermatozoa obtained by TESE for ICSI in patients with testicular cancer and other severe diseases. 15 , 16
In case 1, the patient's semen was cryopreserved prior to chemotherapy. If the patient continued treatment using high volumes of drugs for collagen disease, his doctor worried about the possibility of developing lung cancer, therefore ejaculated sperm was frozen. In case 2, we used fresh ejaculated sperm after surgery prior to chemotherapy. In case 3, we used fresh testicular sperm after surgery and chemotherapy.
The freezing of sperm before chemotherapy and radiation therapy is a simple process. The couple in case 1 particularly benefited from this procedure. In case 2, spermatogenesis partially recovered approximately 6 months postoperatively; thus, this patient did not have his reproductive ability iatrogenically destroyed.
Spermatogenesis is impacted in cancer patients who receive chemotherapy and irradiation therapy. However, sperm karyotypes were studied, 17 using the human sperm‐hamster oocyte system in four patients with testicular cancer and the results in cancer patients were not significantly different than those from control donors. In another study, it was demonstrated that cryopreservation of human sperm does not affect the frequency of numerical or structural chromosomal anomalies or the XY ratio of sperm. 18
As noted by Chan et al., the severity and duration of spermatogenesis impairment after chemotherapy correlates with the number of type A spermatogonia that are destroyed. 19 One matter of concern is the potential genetic risk associated with the use of ART in cancer survivors who have non‐obstructive azoospermia. Genetic counseling is particularly important in this subpopulation regarding both the possibility of an increase in the incidence of tumors in the offspring and the mutagenic effects of chemotherapy on germ cells. 20 However, genetic damage to germ cells from cytotoxic drugs on germ cells is currently controversial. An increased incidence of sperm chromosomal abnormalities has been found in cancer patients during and immediately after receiving chemotherapy, but this is considered to be a temporary effect of cytotoxic drugs. 21 , 22
To date, the clinical impact of a rise in sperm chromosomal abnormalities is not well defined, but it could result in an increase in spontaneous abortions, stillbirths, or birth defects. 23 The published reports contains only a few articles describing successful pregnancy outcomes after TESE and ICSI in patients with persistent non‐obstructi azoospermia following chemotherapy. 19 , 20 , 21 , 22 , 23 , 24
In case 3, although the patient suffered surgical damage, the fecundity of sperm was maintained by using testicular sperm.
We stored the participant's semen who were desirous of future ART (data not shown). The samples were naturally ejaculated semen specimens obtained prior to chemotherapy and irradiation therapy. Currently, many patients diagnosed with severe disease desire to store their semen for ART. At present, it is a comparatively simple matter for us to comply with their request; however, the possibility exists that the owner of our clinic might discontinue the ART program. We recommend that frozen sperm be controlled by a public or governmental organization.
In conclusion, we suggest patients with severe diseases, such as cancer, should store semen before undergoing therapy. For patients who do not have sperm frozen before therapy and develop permanent azoospermia, testicular spermatozoa can be successfully retrieved for ICSI. We actively froze patient's sperm prior to chemotherapy; however, two of three cases used fresh sperm. Our approach can result in pregnancy and the delivery of healthy children; however, informed consent must be given to apprise patients of the potential genetic risk of using frozen sperm from cancer patients.
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