Successful pregnancy after intracytoplasmic sperm injection with testicular spermatozoa transported only under refrigeration
This case report describes two successful pregnancies after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa that were transported under refrigeration.
Two first-time couples consulted our clinic concerned about their primary infertility. No sperm were present in the semen samples from either of the husbands and they were referred to the urology department (UD) of a neighbouring hospital. At the UD, seminiferous tubules were obtained by testicular sperm extraction. The tissue samples were put in a centrifuge tube with phosphate-buffered saline at 6°C and placed with refrigerant in a cushioned styrofoam box that was then transported to our clinic. Immediately upon arrival at our clinic, testicular spermatozoa were extracted. On the same day, ovum pickup was performed and mature oocytes were extracted that were then inseminated by conventional ICSI. Fertilized eggs were cultured for 2 days, and then cleaved embryos were cryopreserved. In one case after 4 months and in the other case after 2 months of cryopreservation, the frozen-thawed embryos were transferred.
Both patients became pregnant and normal, healthy babies were born.
These results suggest that cases of obstructive azoospermia can be treated with ICSI by refrigerated transport of the seminiferous tubules, in cooperation with a UD, in a small single departmental obstetrics and gynecology clinic.
KeywordsIntracytoplasmic sperm injection Obstructive azoospermia Refrigerated transport Seminiferous tubules Testicular sperm extraction
Azoospermia, the absence of sperm in ejaculated semen, is an infrequent cause of male factor infertility that accounts for approximately 5% of infertility in couples . Since testicular sperm extraction (TESE) [2, 3] and microsurgical epididymal sperm aspiration (MESE)  were introduced in the 1990s, infertility treatments using testicular or epididymal spermatozoa have been used as a treatment for azoospermia.
Early in the 1950s, Bunge and Sherman  reported a successful pregnancy after artificial insemination using frozen-thawed sperm. New methods were subsequently developed to preserve the minimum number of spermatozoa needed for ovum fertilization, such as preserving spermatozoa in the zona pellucida where cellular material is removed  and using a cryoloop technique . Some cases of pregnancy after intracytoplasmic sperm injection (ICSI) using frozen-thawed testicular spermatozoa or epididymal spermatozoa have been reported [8, 9, 10]. Thus, TESE–ICSI and MESA–ICSI also became feasible for use by in vitro fertilization clinics in cooperation with male infertility clinics. Moreover, Jaskey and Cohen  reported that sperm could be preserved in test-yolk buffer under refrigeration for 96 h, and Johnson et al.  reported that the buffer actually enhanced the fertilizing capacity of the sperm.
Because we did not have the equipment for TESE at our facility, we carried out TESE–ICSI in cooperation with the urology department (UD) of a neighbouring hospital. Here, we report two cases where TESE was carried out for obstructive azoospermia. The seminiferous tubules were soaked in phosphate-buffered saline (PBS) at 6°C and transported under refrigeration from the UD to our clinic. Spermatozoa in the seminiferous tubules were used for ICSI, and both the women became pregnant.
Subjects, materials, and methods
Preparation of PBS
PBS was prepared by dissolving 2.4 g of Dulbecco’s PBS (−) (Nissui Pharmaceutical, Tokyo, Japan) in 250 mL of Otsuka distilled water (Otsuka Pharmaceutical, Tokyo, Japan), and the solution was then sterilized in an autoclave.
Two first-time couples consulted our clinic because of primary infertility. Because neither woman showed a clear cause of infertility, the semen samples of both men were analyzed. No sperm were present in either sample and the husbands were referred to the UD of a neighbouring hospital, where they were diagnosed with obstructive azoospermia.
The women were treated with the gonadotropin-releasing hormone analogue buserelin acetate (Fuji Pharmaceutical, Tokyo, Japan) and human menopausal gonadotropin (Aska Pharmaceutical, Tokyo, Japan), using a long treatment protocol. When two or more follicles had grown to 18–20 mm in diameter (determined by transvaginal ultrasound), 5000 IU of human chorionic gonadotropin (hCG; Fuji Pharmaceutical) was administered. Ovum pickup (OPU) was performed 35 h after hCG injection. The oocytes were freed from cumulus cells using 0.025% hyaluronidase (Sigma–Aldrich, St. Louis, MO, USA) dissolved in Universal IVF medium (MediCult, Jyllinge, Denmark). Metaphase II oocytes were then cultured for 2 h or more. The oocytes were inseminated by conventional ICSI  in Universal IVF Medium 3 h after OPU. The following morning, fertilization was confirmed by the presence and number of pronuclei and polar bodies, and the fertilized eggs were cultured in Blast Assist System (MediCult) until day 2.
A woman and her husband (both 32 years old) consulted our clinic. In the first ICSI cycle, testicular spermatozoa were cryopreserved after TESE at the UD and transported to our clinic the day before OPU. OPU was performed 13 days after TESE and five mature oocytes were obtained (on cycle day 11). The frozen testicular spermatozoa were thawed and used for ICSI. The following morning, fertilization was confirmed in two eggs. On day 2, a 3-cell embryo and a 2-cell embryo were transferred to the woman’s uterus. For luteal support during the cycle, the patient took 2 mg chlormadinone acetate (Lutoral; Shionogi, Osaka, Japan) three times a day from the time of embryo transfer until the urine hCG test, but no implantation occurred.
In the second ICSI cycle, nine mature oocytes were obtained (on cycle day 13), and the oocytes were inseminated by conventional ICSI .
The following morning, fertilization was confirmed in eight of the oocytes, and the fertilized eggs were cultured until day 2. A 4-cell embryo and a 2-cell embryo were transferred to the patient’s uterus on day 2, but no implantation occurred. For luteal support during the fresh embryo transfer cycle, the patient took an 81 mg acetylsalicylic acid (Bufferin combination tablet A81; Lion, Tokyo, Japan) and a 200 mg progesterone vaginal suppository was inserted once a day from the day following OPU until the urine hCG test. The progesterone vaginal suppositories were made by dissolving progesterone (Sigma–Aldrich) and Macrogol 6000 (Nikko Pharmaceutical, Gifu, Japan) in Macrogol 400 (Yoshida Pharmaceutical, Tokyo, Japan) .
The remaining six embryos were cryopreserved on day 2 using the cryotop technique, as described by Kuwayama .
On the same day as the OPU, the seminiferous tubules were obtained by TESE at the UD. The tissue samples (2 mm3) were placed in a 15-ml centrifuge tube (Sarstedt, Nümbrecht, Germany) with 0.5 ml PBS (the minimum volume that completely soaked the samples) at 6°C and placed in a cushioned styrofoam box (20.7 cm long, 12.5 cm wide, 12.5 cm deep) that contained a refrigerant. The styrofoam box was transported from the UD to our clinic in about 1.5 h. Immediately upon arrival, the tissue samples were minced with scissors, and testicular spermatozoa were extracted. The sperm suspension and minced tissue samples were mixed by pipette with PBS used for transport. The spermatozoa used for ICSI were preserved in PBS under refrigeration until the termination of ICSI. Although 0–1 spermatozoa were motile in all fields of a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel), the progressive motility of the spermatozoa was poor in all fields of the counting chamber and micromanipulator.
Two months after cryopreservation, two frozen cleaved embryos were thawed by the procedure described by Kuwayama , except that thawing was performed at room temperature. Two 4-cell embryos were cultured for 2 days, and a morula and a 5-cell embryo were then transferred to the woman’s uterus. For luteal support during the first frozen embryo transfer cycle, the patient was injected with 25 mg progesterone (Progehormon; Mochida Pharmaceutical, Tokyo, Japan) once every 3 days and she took 2 mg Lutoral three times a day from ovulation until the urine hCG test, but no implantation occurred.
Four months after cryopreservation, two frozen-thawed 3-cell embryos were cultured for 1 day, and two 5-cell embryos were once again transferred. For luteal support during the second frozen embryo transfer cycle, the patient took 2 mg Lutoral three times a day from ovulation until the urine hCG test and she then took dydrogesterone (Duphaston; Daiichi Sankyo, Tokyo, Japan) from the day of the urine hCG test until the fetal heartbeat was verified. Pregnancy was confirmed by a positive urine hCG test 11 days after embryo transfer, and the gestational sac and fetal heartbeat were observed by transvaginal ultrasound 28 days after embryo transfer. No structural abnormalities were observed by ultrasound during any pregnancy checkups. A normal, healthy girl (3340 g) was born at 40 weeks, and she was healthy when observed during the second and fourth weeks after birth.
A 30-year-old woman and her 27-year-old husband consulted our clinic. In the first ICSI cycle, ovarian stimulation, OPU, ICSI, TESE, and transport of seminiferous tubules were performed using the same methods as those described above for Case 1.
Although 3 spermatozoa were motile in all fields of a Makler counting chamber, the progressive motility of the spermatozoa was poor in all fields of the counting chamber and micromanipulator; cytoplasmic droplets extended along the length of the midpiece in most motile spermatozoa. Nine mature oocytes were obtained (on cycle day 14) and four were fertilized. They were cultured until day 2 and cryopreserved. Two months after cryopreservation, two frozen-thawed 4-cell embryos were cultured for 2 days, and a morula and a 4-cell embryo were then transferred to the woman’s uterus. For luteal support during the frozen embryo transfer cycle, the patient inserted a 200 mg progesterone vaginal suppository once a day and she took 2 mg Lutoral three times a day from ovulation until the gestational sac was verified.
Pregnancy was confirmed with a positive urine hCG test 12 days after embryo transfer, and the gestational sac and fetal heartbeat were observed by transvaginal ultrasound 32 days after embryo transfer. No structural abnormalities were observed by ultrasound during pregnancy checkups. A normal, healthy girl (3355 g) was born at 39 weeks, and she was healthy when observed during the second and fourth weeks after birth.
Our results demonstrate how seminiferous tubules soaked in PBS and transported under refrigeration can be used for ICSI after extracting the testicular spermatozoa, with the procedure resulting in successful pregnancies in two patients. This transport method does not require new capabilities, and there is minimal disparity in the technical abilities required for this method and for the transport of frozen spermatozoa because the method is relatively simple; the tissue samples are soaked in PBS without freezing. Moreover, accessibility was simpler, and the cost of refrigerated transport was lower than the cost of transporting frozen spermatozoa, because neither a dry shipper nor liquid nitrogen was needed.
Despite establishing frozen TESE–ICSI, we decided to perform fresh TESE–ICSI on the same day as OPU because of known risks such as transportation delays due to traffic conditions or failure to harvest testicular spermatozoa or oocytes. We attempted this procedure to reduce damage to the testicular spermatozoa. In Case 1, in the first ICSI cycle in this report, frozen TESE–ICSI was performed and two cleaved embryos were transferred, but no implantation occurred.
Several studies have compared the pregnancy rates of fresh sperm-injected eggs to those of frozen sperm-injected eggs. One report showed no significant difference in the implantation rates between oocytes injected with fresh or frozen testicular spermatozoa (implantation rates using fresh/motile sperm were 14%; fresh/immotile, 9%; frozen/motile, 7%; and frozen/immotile, 8%) . De Oliveira et al.  also reported no significant differences when comparing fertilization, pregnancy per patient, and take-home babies achieved using immotile (fresh and frozen-thawed) versus motile (fresh and frozen-thawed) sperm. In addition, they stated that the reliability of the mechanical touch technique was indirectly shown by their results. In contrast, another report showed that implantation and live birth rates per transferred embryo were significantly lower after ICSI with frozen-thawed spermatozoa (9.1%) than those with fresh testicular spermatozoa (24.6%) .
For treatments in which testicular spermatozoa are frozen, cryoprotective agents easily permeate the spermatozoa because of their small size, and spermatozoa are probably affected by the toxicity of the agent. Spermatozoa were unharmed by our method because no cryoprotective agent was used. Some reported frozen-thawed sperm cryoinjuries include membrane disruption, diminished motility, and deterioration of viability , as well as DNA damage . Dondero et al.  reported no significant differences when comparing motility or hypoosmotic viability test recoveries using frozen versus refrigerated sperm from normospermic subjects for 48 h, but in infertile subjects, a significant decrease in the percentage recovery of straight progressive motility and the hypoosmotic viability test was observed in cryopreserved sperm compared to those refrigerated for 48 h. Another advantage of our procedure is also that cryoinjuries to spermatozoa would be reduced.
Van Thuan et al.  reported that sperm activation competence was prolonged by increasing the bovine serum albumin concentration from 0 to 4 mg/ml in potassium simplex optimized medium supplemented with amino acids (KSOMaa), which is used for preservation at room temperature. Therefore, we considered whether to use refrigeration or culture media containing protein. However, in this study, we excluded the components not normally found in organisms such as antibiotic, egg yolk, and so on, because preservation was necessary for only a few hours.
Several studies have reported the preservation of epididymides by using mineral oil under refrigeration [21, 22]. We considered that placing the seminiferous tubules in mineral oil would make them resistant to temperature change. However, the mineral oil was removed from the tissue samples before they were used for ICSI. We decided to use PBS because a decision was made to perform ICSI as soon as the samples reached our clinic.
Several studies on the preservation of sperm under refrigeration have reported optimum temperature control of 4–6°C [11, 12, 22, 23, 24, 25]. Furthermore, one study reported that the transportation of C57BL/6 mouse strain epididymides was more successful at 7°C than at 5°C . We decided on a temperature control of 6°C because taking the findings of these studies [11, 12, 22, 23, 24, 25] into consideration offered the best chance for success.
In the present report, testicular spermatozoa were preserved in refrigerated PBS for about 2.5–3 h, which was the duration required to remove the seminiferous tubules at the end of ICSI. Therefore, we were unable to confirm whether testicular spermatozoa preserved under the same conditions at 3 h or more would retain fertility. However, in cases of obstructive azoospermia, such as the two cases in the present study, this result showed that the simple transport of testicular spermatozoa soaked in PBS under refrigeration was possible for short time periods. These results suggest that cases of obstructive azoospermia can be treated with ICSI by using refrigerated transport of the seminiferous tubules in cooperation with the UD at a small single obstetrics and gynecology departmental clinic.
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