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Birth of a healthy infant after intracytoplasmic sperm injection using pentoxifylline-activated sperm from a patient with Kartagener’s syndrome
CASE REPORT Birth of a healthy infant after intracytoplasmic sperm injection using pentoxifylline-activated sperm from a patient with Kartagener’s syndrome Hiromitsu Hattori, B.S.,a Yukiko Nakajo, B.S.,a Chizuru Ito, M.D., Ph.D.,b Yoshiro Toyama, D.V.M., Ph.D.,b Kiyotaka Toshimori, M.D., Ph.D.,b and Koichi Kyono, M.D., Ph.D.a a
Kyono ART Clinic, Sendai; and b Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan
Objective: To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartagener’s syndrome. Design: Case report. Setting: Private assisted reproductive technology clinic in Japan. Patient(s): A couple with male factor infertility due to Kartagener’s syndrome. Intervention(s): Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. Main Outcome Measure(s): Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. Result(s): The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. Conclusion(s): With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartagener’s syndrome. (Fertil Steril 2011;95:2431.e9–e11. 2011 by American Society for Reproductive Medicine.) Key Words: Kartagener’s syndrome, immotile cilia, pentoxifylline, ICSI, vitrification
Immotile cilia syndrome is an autosomal recessive disorder characterized by defective cilial ultrastructure in ciliated cells and affects approximately 1 in 20,000 newborns. Kartagener’s syndrome (KS) is characterized by the classic triad of chronic sinusitis, bronchiectasis, and situs inversus (1). The dynein arms connecting the microtubules are shortened or absent, which results in sperm immobility and ciliary epithelial dysfunction (2, 3). Therefore, male patients are invariably infertile because of immotile sperm. To date, the literature contains articles on successful fertilization and pregnancy using sperm from patients with KS (3–9). In this report, we describe a successful pregnancy outcome that was achieved using ejaculated immotile sperm from a patient with KS. Intracytoplasmic sperm injection (ICSI) was used, with ejaculated pentoxifylline-activated sperm.
CASE REPORT In December 2009, a couple referred to our clinic expressed a desire to achieve a pregnancy. The woman, aged 30 years, and her husband, Received March 2, 2011; accepted March 14, 2011; published online April 20, 2011. H.H. has nothing to disclose. Y.N. has nothing to disclose. C.I. has nothing to disclose. Y.T. has nothing to disclose. K.T. has nothing to disclose. K.K. has nothing to disclose. Reprint requests: Koichi Kyono, M.D., Ph.D., Kyono ART Clinic, Mitsui-Seimei, Sendai Honcho Bldg. 3F, 1-1-1 Honcho, Aoba-ku, Sendai 980-0014, Japan (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.03.074
aged 31 years, had been trying to achieve a pregnancy for 1 year. The results of the woman’s diagnostic workup (including hormonal evaluations, hysterosalpingography, hysteroscopy, and transvaginal ultrasonography) were within normal limits. The husband exhibited normal secondary sex characteristics, and both testicular volumes were normal. His serum FSH, LH, total T, and PRL levels were all within normal limits. His history and physical examination exhibited classic finding for KS: chronic bronchitis, sinusitis, dysosmia, and dextrocardia. Semen analysis revealed ejaculated volumes ranging from 1.2 mL to 5.7 mL and concentrations from 6.2 106/mL to 8.4 106/mL with severe oligozoospermia (World Health Organization 2010 criteria); furthermore, all the sperm observed on several fields in a Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel) exhibited no motility on several fields. Eosin-Y stain revealed a viability of 54%. Sperm morphology was observed by transmission electron microscopy (Fig. 1). The tails exhibited an absence of one or both inner and outer dynein arms, which included an A subunit and a B subunit (Fig. 1C and D). Almost all of the sperm contained an excessive fibrous sheath and an irregular rib arrangement (Fig. 1C and D); furthermore, the central pair was absent, and the 9þ2 axonemal structure was incomplete (Fig. 1C and D). Institutional review board approval was not required to proceed with therapy. In December 2009, ovarian stimulation was conducted via the GnRH antagonist method. An injection of 5,000 U of hCG
Fertility and Sterility Vol. 95, No. 7, June 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Electron micrographs of the sperm. Abnormal tails are shown. (A and B) Low-magnification micrographs showing disarranged tail components of the middle piece and principal piece. The heads are normal in structure. (C and D) Cross-sections of the principal pieces. Central pairs of the axoneme are lacking. Most outer dynein arms and a few inner dynein arms are lacking. Excess fibrous sheaths are observed. Scale bars: 1 mm in A and B, 0.1 mm in C and D.
Hattori. Activated sperm in Kartagener’s syndrome. Fertil Steril 2011.
(Gonatropin; ASUKA Pharmaceutical, Tokyo, Japan) was administered when the dominant follicle reached a mean diameter of 20 mm. Vaginal ultrasound-guided follicle puncture was conducted 36 hours after the hCG injection. Fourteen oocytes were retrieved, and 12 metaphase II oocytes were obtained. The sperm concentration was 0.9 106/mL, with no motility and 83.3% abnormal morphology. The sperm were centrifuged and washed twice with human tuba1 fluid medium plus 10% albumin (Irvine Scientific, Irvine, CA). All sperm were immotile on several light microscopic fields; therefore, pentoxifylline was added to the sperm medium at a final concentration of 1 mg/mL. After 10 minutes of incubation, a few sperm exhibited slight progressive movement. The slightly motile sperm were transferred to a polyvinylpirrolidone drop by manipulation and washed several times to remove the pentoxifylline; subsequently, ICSI was performed using these activated motile sperm. To obtain the two-pronuclei stage, injected oocytes were incubated overnight at 37 C in an atmosphere of 6% CO2, 5% O2, and 89% N2 under humidified conditions. Seven oocytes were fertilized, and four of the seven embryos developed into blastocysts. To avoid ovarian hyperstimulation syndrome, all blastocysts were cryopreserved by the vitrification method described by Kyono et al. (10). Two months later, one vitrified blastocyst (Gardner’s criteria; 4AB) was warmed and transferred on cycle day 20, which was the fifth day after injection of 12 mg P per day (Shionogi, Osaka, Japan). The luteal phase was supported by oral P administration (6 mg three times daily). A positive serum b-hCG (266.0 mIU/mL) was obtained 14 days later; at 7 weeks and 4 days of gestation a fetal heartbeat was visualized on ultrasound. Fortunately, the patient maintained good health throughout her pregnancy. Ultimately, a healthy female infant was delivered in November 2010 at 40 weeks’ gestation; the weight was 2,736 g, and no congenital abnormalities were noted.
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Hattori et al.
DISCUSSION To date, varying degrees of sperm motility have been reported in patients with KS; however, most patients with KS are reported to have 100% immotile sperm in their ejaculate (1, 4–9, 11). Some articles have reported a successful pregnancy and delivery after ICSI using ejaculated or testicular extracted sperm (4, 7, 8). In this case, no motile sperm were found in the ejaculated specimen. Men with immotile cilia syndrome together with situs inversus and bronchiectasis are referred to as having KS (1). Afzeliu et al. (12) were the first to recognize that the relationship of KS to male infertility was due to immotile sperm. The sperm tail has dynein arms, which link microtubular doublets in the axonemal complex (13). These dynein arms are the site of adenosine triphosphatase activity and mediate the alignment between doublet microtubules, essential for normal ciliary motion (14). When dynein arms are lacking, a variety of ineffective ciliary motion patterns can occur, resulting in poor or no sperm motility. We perceived that pentoxifylline is a nonselective phosphodiesterase inhibitor, which inhibits the breakdown of cyclic adenosine monophosphate to adenosine monophosphate (15). It is theorized that a few sperm with normal morphology positively reacted to pentoxifylline despite the finding that almost all sperm exhibited abnormal morphology on electron microscopy. When we conducted ICSI, we searched for sperm with nearly normal morphology and used these sperm for intracytoplasmic morphologically selected sperm injection. In view of our results, it seems that sperm activation of pentoxifylline may improve the motility in patients with KS. In summary, we report a successful pregnancy outcome after the transfer of a single vitrified–warmed blastocyst, which was fertilized via ICSI with pentoxifylline-activated sperm from a patient with KS.
Activated sperm in Kartagener’s syndrome
Vol. 95, No. 7, June 2011
REFERENCES 1. Afzelius BA. The immotile-cilia syndrome: a microtubule-associated defect. CRC Crit Rev Biochem 1985;19:63–87. 2. Palmblad J, Mossberg B, Afzelius BA. Ultrastructural, cellular, and clinical features of the immotilecilia syndrome. Annu Rev Med 1984;35:481–92. 3. Yokota T, Ohno N, Tamura K, Seita M, Toshimori K. Ultrastructure and function of cilia and spermatozoa flagella in a patient with Kartagener’s syndrome. Intern Med 1993;32:593–7. 4. Kaushal M, Baxi A. Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener’s cilia syndrome. Fertil Steril 2007;88:497.e9–11. 5. Kay VJ, Irvine DS. Successful in vitro fertilization pregnancy with spermatozoa from a patient with Kartagener’s syndrome. Hum Reprod 2000;15: 135–8. 6. von Zumbusch A, Fiedler K, Mayerhofer A, Jessberger B, Ring J, Vogt HJ. Birth of healthy chil-
Fertility and Sterility
7.
8.
9.
10.
dren after intracytoplasmic sperm injection in two couples with male Kartagener’s syndrome. Fertil Steril 1998;70:643–6. Cayan S, Conaghan J, Schriock ED, Ryan IP, Black LD. Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener/ immotile cilia syndrome. Fertil Steril 2001;76:612–4. Westlander G, Barry M, Petrucco O, Norman R. Different fertilization rates between immotile testicular spermatozoa and immotile ejaculated spermatozoa for ICSI in men with Kartagener’s syndrome: case reports. Hum Reprod 2003;8:1286–8. Matsumoto Y, Goto S, Hashimoto H, Kokeguchi S, Shiotani M, Okada H. A healthy birth after intracytoplasmic sperm injection using ejaculated spermatozoa from a patient with Kartagener’s syndrome. Fertil Steril 2010;93:2074.e17–9. Kyono K, Nakajo Y, Kumagai S, Nishinaka C. Vitrifying and warming of oocytes using cryotop. In: Tucker MJ, Liebermann J, editors. Vitrification
11.
12.
13.
14. 15.
in assisted reproduction: a user’s manual and trouble-shooting guide. London: Informa UK; 2007. p. 153–61. Abu-Musa A, Hannoun A, Khabbaz A, Devroey P. Failure of fertilization after intracytoplasmic sperm injection in a patient with Kartagener’s syndrome and totally immotile spermatozoa. Hum Reprod 1999;14:2517–8. Afzelius BA, Eliasson R, Johnson O, Lindholmer C. Lack of dynein arms in immotile human spermatozoa. J Cell Biol 1975;66:225. Eliasson R, Mossberg B, Camner P, Afzelius BA. The immotile-cilia syndrome. A congenital ciliary abnormality as an etiologic factor in chronic airway infections and male sterility. N Engl J Med 1997;1:297. Afzelius BA. Disorders of ciliary motility. Hosp Prac (Off Ed) 1986;15:63–87. Tash JS, Means AR. Cyclic adenosine 30 ,50 monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod 1983;28:75–104.
2431.e11
Kyono ART Clinic, Sendai; and b Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan
Objective: To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartagener’s syndrome. Design: Case report. Setting: Private assisted reproductive technology clinic in Japan. Patient(s): A couple with male factor infertility due to Kartagener’s syndrome. Intervention(s): Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. Main Outcome Measure(s): Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. Result(s): The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. Conclusion(s): With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartagener’s syndrome. (Fertil Steril 2011;95:2431.e9–e11. 2011 by American Society for Reproductive Medicine.) Key Words: Kartagener’s syndrome, immotile cilia, pentoxifylline, ICSI, vitrification
Immotile cilia syndrome is an autosomal recessive disorder characterized by defective cilial ultrastructure in ciliated cells and affects approximately 1 in 20,000 newborns. Kartagener’s syndrome (KS) is characterized by the classic triad of chronic sinusitis, bronchiectasis, and situs inversus (1). The dynein arms connecting the microtubules are shortened or absent, which results in sperm immobility and ciliary epithelial dysfunction (2, 3). Therefore, male patients are invariably infertile because of immotile sperm. To date, the literature contains articles on successful fertilization and pregnancy using sperm from patients with KS (3–9). In this report, we describe a successful pregnancy outcome that was achieved using ejaculated immotile sperm from a patient with KS. Intracytoplasmic sperm injection (ICSI) was used, with ejaculated pentoxifylline-activated sperm.
CASE REPORT In December 2009, a couple referred to our clinic expressed a desire to achieve a pregnancy. The woman, aged 30 years, and her husband, Received March 2, 2011; accepted March 14, 2011; published online April 20, 2011. H.H. has nothing to disclose. Y.N. has nothing to disclose. C.I. has nothing to disclose. Y.T. has nothing to disclose. K.T. has nothing to disclose. K.K. has nothing to disclose. Reprint requests: Koichi Kyono, M.D., Ph.D., Kyono ART Clinic, Mitsui-Seimei, Sendai Honcho Bldg. 3F, 1-1-1 Honcho, Aoba-ku, Sendai 980-0014, Japan (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.03.074
aged 31 years, had been trying to achieve a pregnancy for 1 year. The results of the woman’s diagnostic workup (including hormonal evaluations, hysterosalpingography, hysteroscopy, and transvaginal ultrasonography) were within normal limits. The husband exhibited normal secondary sex characteristics, and both testicular volumes were normal. His serum FSH, LH, total T, and PRL levels were all within normal limits. His history and physical examination exhibited classic finding for KS: chronic bronchitis, sinusitis, dysosmia, and dextrocardia. Semen analysis revealed ejaculated volumes ranging from 1.2 mL to 5.7 mL and concentrations from 6.2 106/mL to 8.4 106/mL with severe oligozoospermia (World Health Organization 2010 criteria); furthermore, all the sperm observed on several fields in a Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel) exhibited no motility on several fields. Eosin-Y stain revealed a viability of 54%. Sperm morphology was observed by transmission electron microscopy (Fig. 1). The tails exhibited an absence of one or both inner and outer dynein arms, which included an A subunit and a B subunit (Fig. 1C and D). Almost all of the sperm contained an excessive fibrous sheath and an irregular rib arrangement (Fig. 1C and D); furthermore, the central pair was absent, and the 9þ2 axonemal structure was incomplete (Fig. 1C and D). Institutional review board approval was not required to proceed with therapy. In December 2009, ovarian stimulation was conducted via the GnRH antagonist method. An injection of 5,000 U of hCG
Fertility and Sterility Vol. 95, No. 7, June 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
2431.e9
FIGURE 1 Electron micrographs of the sperm. Abnormal tails are shown. (A and B) Low-magnification micrographs showing disarranged tail components of the middle piece and principal piece. The heads are normal in structure. (C and D) Cross-sections of the principal pieces. Central pairs of the axoneme are lacking. Most outer dynein arms and a few inner dynein arms are lacking. Excess fibrous sheaths are observed. Scale bars: 1 mm in A and B, 0.1 mm in C and D.
Hattori. Activated sperm in Kartagener’s syndrome. Fertil Steril 2011.
(Gonatropin; ASUKA Pharmaceutical, Tokyo, Japan) was administered when the dominant follicle reached a mean diameter of 20 mm. Vaginal ultrasound-guided follicle puncture was conducted 36 hours after the hCG injection. Fourteen oocytes were retrieved, and 12 metaphase II oocytes were obtained. The sperm concentration was 0.9 106/mL, with no motility and 83.3% abnormal morphology. The sperm were centrifuged and washed twice with human tuba1 fluid medium plus 10% albumin (Irvine Scientific, Irvine, CA). All sperm were immotile on several light microscopic fields; therefore, pentoxifylline was added to the sperm medium at a final concentration of 1 mg/mL. After 10 minutes of incubation, a few sperm exhibited slight progressive movement. The slightly motile sperm were transferred to a polyvinylpirrolidone drop by manipulation and washed several times to remove the pentoxifylline; subsequently, ICSI was performed using these activated motile sperm. To obtain the two-pronuclei stage, injected oocytes were incubated overnight at 37 C in an atmosphere of 6% CO2, 5% O2, and 89% N2 under humidified conditions. Seven oocytes were fertilized, and four of the seven embryos developed into blastocysts. To avoid ovarian hyperstimulation syndrome, all blastocysts were cryopreserved by the vitrification method described by Kyono et al. (10). Two months later, one vitrified blastocyst (Gardner’s criteria; 4AB) was warmed and transferred on cycle day 20, which was the fifth day after injection of 12 mg P per day (Shionogi, Osaka, Japan). The luteal phase was supported by oral P administration (6 mg three times daily). A positive serum b-hCG (266.0 mIU/mL) was obtained 14 days later; at 7 weeks and 4 days of gestation a fetal heartbeat was visualized on ultrasound. Fortunately, the patient maintained good health throughout her pregnancy. Ultimately, a healthy female infant was delivered in November 2010 at 40 weeks’ gestation; the weight was 2,736 g, and no congenital abnormalities were noted.
2431.e10
Hattori et al.
DISCUSSION To date, varying degrees of sperm motility have been reported in patients with KS; however, most patients with KS are reported to have 100% immotile sperm in their ejaculate (1, 4–9, 11). Some articles have reported a successful pregnancy and delivery after ICSI using ejaculated or testicular extracted sperm (4, 7, 8). In this case, no motile sperm were found in the ejaculated specimen. Men with immotile cilia syndrome together with situs inversus and bronchiectasis are referred to as having KS (1). Afzeliu et al. (12) were the first to recognize that the relationship of KS to male infertility was due to immotile sperm. The sperm tail has dynein arms, which link microtubular doublets in the axonemal complex (13). These dynein arms are the site of adenosine triphosphatase activity and mediate the alignment between doublet microtubules, essential for normal ciliary motion (14). When dynein arms are lacking, a variety of ineffective ciliary motion patterns can occur, resulting in poor or no sperm motility. We perceived that pentoxifylline is a nonselective phosphodiesterase inhibitor, which inhibits the breakdown of cyclic adenosine monophosphate to adenosine monophosphate (15). It is theorized that a few sperm with normal morphology positively reacted to pentoxifylline despite the finding that almost all sperm exhibited abnormal morphology on electron microscopy. When we conducted ICSI, we searched for sperm with nearly normal morphology and used these sperm for intracytoplasmic morphologically selected sperm injection. In view of our results, it seems that sperm activation of pentoxifylline may improve the motility in patients with KS. In summary, we report a successful pregnancy outcome after the transfer of a single vitrified–warmed blastocyst, which was fertilized via ICSI with pentoxifylline-activated sperm from a patient with KS.
Activated sperm in Kartagener’s syndrome
Vol. 95, No. 7, June 2011
REFERENCES 1. Afzelius BA. The immotile-cilia syndrome: a microtubule-associated defect. CRC Crit Rev Biochem 1985;19:63–87. 2. Palmblad J, Mossberg B, Afzelius BA. Ultrastructural, cellular, and clinical features of the immotilecilia syndrome. Annu Rev Med 1984;35:481–92. 3. Yokota T, Ohno N, Tamura K, Seita M, Toshimori K. Ultrastructure and function of cilia and spermatozoa flagella in a patient with Kartagener’s syndrome. Intern Med 1993;32:593–7. 4. Kaushal M, Baxi A. Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener’s cilia syndrome. Fertil Steril 2007;88:497.e9–11. 5. Kay VJ, Irvine DS. Successful in vitro fertilization pregnancy with spermatozoa from a patient with Kartagener’s syndrome. Hum Reprod 2000;15: 135–8. 6. von Zumbusch A, Fiedler K, Mayerhofer A, Jessberger B, Ring J, Vogt HJ. Birth of healthy chil-
Fertility and Sterility
7.
8.
9.
10.
dren after intracytoplasmic sperm injection in two couples with male Kartagener’s syndrome. Fertil Steril 1998;70:643–6. Cayan S, Conaghan J, Schriock ED, Ryan IP, Black LD. Birth after intracytoplasmic sperm injection with use of testicular sperm from men with Kartagener/ immotile cilia syndrome. Fertil Steril 2001;76:612–4. Westlander G, Barry M, Petrucco O, Norman R. Different fertilization rates between immotile testicular spermatozoa and immotile ejaculated spermatozoa for ICSI in men with Kartagener’s syndrome: case reports. Hum Reprod 2003;8:1286–8. Matsumoto Y, Goto S, Hashimoto H, Kokeguchi S, Shiotani M, Okada H. A healthy birth after intracytoplasmic sperm injection using ejaculated spermatozoa from a patient with Kartagener’s syndrome. Fertil Steril 2010;93:2074.e17–9. Kyono K, Nakajo Y, Kumagai S, Nishinaka C. Vitrifying and warming of oocytes using cryotop. In: Tucker MJ, Liebermann J, editors. Vitrification
11.
12.
13.
14. 15.
in assisted reproduction: a user’s manual and trouble-shooting guide. London: Informa UK; 2007. p. 153–61. Abu-Musa A, Hannoun A, Khabbaz A, Devroey P. Failure of fertilization after intracytoplasmic sperm injection in a patient with Kartagener’s syndrome and totally immotile spermatozoa. Hum Reprod 1999;14:2517–8. Afzelius BA, Eliasson R, Johnson O, Lindholmer C. Lack of dynein arms in immotile human spermatozoa. J Cell Biol 1975;66:225. Eliasson R, Mossberg B, Camner P, Afzelius BA. The immotile-cilia syndrome. A congenital ciliary abnormality as an etiologic factor in chronic airway infections and male sterility. N Engl J Med 1997;1:297. Afzelius BA. Disorders of ciliary motility. Hosp Prac (Off Ed) 1986;15:63–87. Tash JS, Means AR. Cyclic adenosine 30 ,50 monophosphate, calcium and protein phosphorylation in flagellar motility. Biol Reprod 1983;28:75–104.
2431.e11