Birth after intracytoplasmic sperminjection with use of testicular sperm frommen with Kartagener/immotile ciliasyndrome

Selahittin Cayan, M.D.,a Joseph Conaghan, Ph.D.,c Eldon D. Schriock, M.D.,c

Isabelle P. Ryan, M.D.,c Lauri D. Black, M.S.,a,b and Paul J. Turek, M.D.a,b

University of California San Francisco School of Medicine, San Francisco, California

Objective: To describe two cases of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)with testicular sperm in men with immotile cilia syndromes.

Design: Case report.

Setting: A university-based male infertility clinic and assisted reproduction unit.

Patient(s): Two couples with male factor infertility due to Kartagener/immotile cilia syndrome.

Intervention(s): IVF/ICSI with testicular sperm.

Main outcome measure(s):Semen characteristics, sperm viability, fertilization rate, and pregnancy.

Result(s): With testicular sperm, the two pronuclear fertilization rates were 63% and 60% in two cases. Onecase resulted in the birth of normal healthy girl.

Conclusion(s): With testicular sperm, successful oocyte fertilization after ICSI in couples with male Kart-agener/immotile cilia syndrome is possible despite the lack of sperm motility. (Fertil Sterilt 2001;76:612–14.©2001 by American Society for Reproductive Medicine.)

Key Words: Testicular sperm, Kartagener syndrome, immotile cilia syndrome, intracytoplasmic sperminjection

Immotile cilia syndromes are characterizedby defective ciliary ultrastructure with abnor-malities or absence of dynein arms or axon-emal microtubules (1). An estimated incidenceof this condition, which affects the sperm tailand the cilia of the respiratory tract, is one in20,000 live births. Clinical manifestations ofimmotile cilia syndrome include chronic pan-sinusitus, recurrent respiratory tract infections,bronchiectasis, and infertility in the male. Kart-agener syndrome, a variant of immotile ciliasyndrome, is predominantly an autosomal re-cessive condition and is characterized by situsinversus in conjunction with immotile cilia (1).

Although immotile cilia syndromes are veryrare, infertility is the rule in men who areaffected. Interestingly, females are usually fer-tile because fallopian tube motility is muscularand not ciliary in nature (2). In afflicted men,ejaculated sperm are generally immotile, butthere are cases in which low or normal sperm

motility has been described. Sperm concentra-tions are normal or even high. The spermatozoafrom patients with these conditions are usuallymorphologically normal on light microscopyand the immotility does not necessarily signifya lack of viability.

The use of in vitro fertilization (IVF) andintracytoplasmic sperm injection (ICSI) to treatmale infertility due to immotile cilia syn-dromes has been reported. However, even withthese sophisticated technologies, very fewpregnancies and births have been achievedwith ejaculated sperm in infertility due to thiscondition (2–4). One reason for low successrates may be that the most commonly usedmethod for selecting viable sperm for ICSI ismotility and this common characteristic ofsperm is absent in affected men. Consequently,the likelihood of selecting a nonviable spermfor ICSI is higher in these cases.

Received May 22, 2000;revised and accepted April26, 2001.Reprint requests: Paul J.Turek, M.D., Department ofUrology, University ofCalifornia San FranciscoSchool of Medicine, 2330Post Street, 6th Floor, SanFrancisco, California94115-1695 (FAX: 415-353-7252; E-mail:mrvas@itsa.ucsf.edu).a Department of Urology,University of California SanFrancisco School ofMedicine.b Department of Obstetrics,Gynecology andReproductive Sciences,University of California SanFrancisco School ofMedicine.c California Pacific FertilityCenter, San Francisco,California.

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We report here the first 2 cases of IVF and ICSI in menwith immotile cilia syndromes that were performed withtesticular sperm as a source of male gamete. We also de-scribe added selection methods to ensure that viable spermwere chosen and report a healthy live birth in one case.

MATERIALS AND METHODS

Case 1The patient was a 43-year-old male with a 31-year-old

healthy gravida zero partner. The couple had attempted toconceive for 21⁄2 years without success before presentationfor medical care. His history and physical examination ex-hibited classic findings for Kartagener syndrome, with re-current pneumonia, chronic sinusitis, bronchiectasis, anddextrocardia. The electron microscopy of the respiratorytract cilia showed completely absent dynein arms. In addi-tion, the patient was being considered for heart–lung trans-plantation. He had normal secondary sex characteristicswithout evidence of gynecomastia. Scrotal examination re-vealed bilaterally descended testes of normal size (20 mL)and soft consistency. The vas deferens was palpable on eachside and there was no palpable varicocele. He had threeunaffected siblings.

Semen evaluation revealed a volume of 0.75 mL with nosperm (Table 1). A centrifuged pellet also contained nosperm. The seminal pH was normal, and the transrectalultrasonography showed normal findings, indicating no evi-dence of ejaculatory duct obstruction. Serum follicle stimu-lating hormone level was 14 mIU/mL (Normal: 2–17), lu-teinizing hormone level was 11 mIU/mL (normal: 4–18),and testosterone level was 460 ng/dL (normal: 260–1,000).A testis biopsy was performed before referral and demon-strated hypospermatogenesis bilaterally.

Before proceeding with IVF and ICSI, the couple under-went genetic counseling for immotile cilia syndrome and foranalysis of genetic issues surrounding testis failure. Thepatient exhibited a normal 46,XY male karyotype and

showed no evidence for Y chromosome microdeletions. In-stitutional review board approval was not required to pro-ceed with therapy.

The couple proceeded with IVF and ICSI using testicularsperm. All sperm extracted from testis tissue were immotilein culture and thus the hypoosmotic swelling (HOS) test wasapplied to identify viable immotile sperm. After extractionfrom the testis tissue by micropipette, individual sperm wereplaced in a solution of 75 mM fructose and 25 mM citrate forseveral seconds, and observed under phase contrast micros-copy for tail coiling within the micropipette. Sperm thatexhibited such tail coiling were considered viable and im-mediately placed into an isolated drop of P1 medium (IrvineScientific, Santa Ana, CA) supplemented with 10% V/Vsynthetic serum substitute (Irvine Scientific, Santa Ana, CA).These sperm were then used for ICSI. From 19 matureoocytes, 12 (63%) showed two-pronuclear fertilization. Twogood quality embryos were transferred back to the uterus,and the remaining 10 were cryopreserved. Although thecouple failed to achieve a pregnancy on this attempt, they didconceive on a subsequent transfer of 3 frozen-thawed em-bryos. This resulted in the birth of a normal healthy girl.

Case 2The patient was a 37-year-old male whose 37-year-old

partner had not been pregnant in the past. The couple hadbeen attempting to initiate a pregnancy for 10 years beforereferral. She had very few risk factors for infertility, and aclomiphene citrate challenge test performed to assess ovar-ian reserve was normal.

The patient had no dextrocardia, but his medical historywas significant for chronic sinusitis and bronchitis withoutpneumonias. On physical examination, he had normal sec-ondary sex characteristics. Testicular volumes were 20 mLon the right and 18 mL on the left, with normal consistency.Both vasa deferentia were palpably normal and the epidid-ymides were not dilated. No varicocele was detected. Digitalrectal examination was normal.

On further testing, serum hormone revealed follicle-stim-ulating hormone (FSH): 4.7 mIU/mL (normal: 1–12), LH:2.8 mIU/mL (Normal:2–12), and testosterone: 504 ng/mL.Semen evaluation showed ejaculate volumes ranging from 1to 4 mL, with sperm concentrations ranging from 4.8 to 24.1million/mL. In no analysis was there evidence of spermmotility (Table 1). Seminal pH was normal, and an indirectantisperm antibody assay (ImmunoSpheres, Bioscreen, Inc.,NY, NY) was negative. An eosin-Y stain of the ejaculateshowed that 5% to 25% of the sperm were viable. Transmis-sion electron microscopy revealed that 2% of all sperm hadnormal dynein arms and a 912 axonemal microtubule mor-phology, and the remaining 98% were missing inner andouter dynein arms within the 912 morphology.

The patient was diagnosed with immotile cilia syndrome.He had three unaffected sisters. The couple underwent ge-

T A B L E 1

Semen parameters in two men with immotile ciliasyndrome.

Semen parameter Case 1 (range) Case 2 (range)

Ejaculate volume (mL) 0.75 1–4pH 7.4 7.2–7.6Sperm count (million/mL) 0 4.8–24.1Sperm motility (%) — 0Forward progression — 0World Health Organization (WHO)

morphology (% normal)— 0

Eosin-Y test (% viable) — 5–25

Cayan. Testis sperm and immotile cilia syndromes. Fertil Steril 2001.

FERTILITY & STERILITY t 613

netic counseling for this condition within the PROGENI™(Program in the Genetics of Infertility, San Francisco, CA)clinic before proceeding with assisted reproduction. Beforereferral, the couple had undergone 3 prior IVF and ICSIcycles with unselected ejaculated sperm. During the firstcycle, 5 eggs were retrieved and no fertilization achieved. Inthe second cycle, 6 eggs were retrieved and one fertilizednormally; one embryo was transferred but no pregnancyresulted. On the third cycle, 6 eggs were retrieved but nonormal fertilization achieved. Institutional review board ap-proval was not required to proceed with therapy.

On the day of IVF oocyte retrieval, the patient providedan ejaculate for ICSI. Sperm morphology was highly abnor-mal with half the sperm exhibiting short tails and the otherhalf coiled tails. Because of such abnormal morphology atbaseline, the hypoosmotic swelling assay was not applied todetermine which sperm were viable. Instead, an eosin-Yvital stain was performed and revealed that,5% of spermwere viable. It was therefore decided to retrieve testicularsperm to increase the likelihood of finding viable sperm forICSI.

After local anesthesia was administered in a spermaticcord block, testis sperm aspiration was performed on theright testicle. Using a 19-gauge needle, 10 to 20 mg of tissuewere retrieved with two needle passes. Examination of thetissue in the IVF laboratory revealed that the testis sperm,although plentiful in number, exhibited the same two mor-phologic variants that were identified in the ejaculate. Incontrast, however, an eosin-Y vital stain performed on thesecells revealed 95% viability by dye exclusion, suggestingthat the vast majority of these sperm were viable. Therefore,the most morphologically normal appearing sperm wereselected from among the unstained testicular cell populationfor ICSI.

At oocyte retrieval, 9 eggs were obtained and 6 had a firstpolar body. ICSI was performed on these 6 oocytes and 4(60%) exhibited normal two-pronuclear formation within 18hours. All 4 embryos were replaced after 72 hours and nopregnancy was achieved.

DISCUSSION

The genetic basis for immotile cilia syndromes is not welldefined. Cellular motility is likely to be controlled by theaction of over 200 distinct genes. Although no single genehas been found to date in this disease, the inheritance patternderived from family pedigrees suggests that it is likely to beautosomal recessive. A related condition, the stumped tailsyndrome, may have as its basis dysplasia of the spermfibrous sheath. It is postulated that this defect arises from theexistence of redundant and disorganized bundles of fibroussheath material, which create an abnormally rigid, exoskel-

etal shell around the axonemal structures and disrupt normalaxonemal growth, stability, and motility (2). Indeed, onepatient in this study may have exhibited these findings.

Pregnancies and births in cases of immotile cilia syn-drome have been reported with ejaculated but not testicularsperm. Kay and Irvine (4) described the first successfulpregnancy with conventional IVF with sperm from a manwith Kartagener syndrome. The male infant conceived hadno evidence of situs invertus. Even with ICSI, very fewcouples with male Kartagener/immotile cilia syndrome havehad successful fertilization and pregnancy success to date.von Zumbusch et al. reported the first two pregnancies incouples with Kartagener syndrome with complete astheno-spermia and electron microscopically confirmed axonemalabnormalities (3). One couple conceived healthy twins (maleand female) and the other a healthy female singleton.Olmedo et al. (2) described ICSI with ejaculated immotilespermatozoa from a man with dysplasia of the fibrous sheathand dynein arm deficiency. From a single ICSI cycle, 3 of 4normally fertilized oocytes were transferred and a healthygirl conceived. In our study in which ICSI is used withtesticular sperm from affected men, both couples achievedexcellent oocyte fertilization rates and one achieved a birth.

It is important to note that normal oocyte fertilizationafter ICSI is not to be assumed in couples with immotile ciliasyndrome. Indeed, severe or strategic defects in sperm ax-onemal structure might preclude normal two-pronuclear de-velopment after ICSI, given the integral importance of thecentriole and sperm microtubular assembly to this process.Clinically, low fertilization rates and even fertilization fail-ures have been observed. In addition to the existence ofsperm-derived defects that preclude normal early embryodevelopment, fertilization failure after ICSI in couples withimmotile cilia syndromes could also be explained by the factthat nonviable sperm are chosen for ICSI. Indeed, the use ofunselected, immotile sperm is usually associated with fairlylow fertilization rates. We attempted to bypass this problemwith viable sperm selection measures such as the HOS assayor by using testis sperm that were generally very viable.Such selection measures were applied to eliminate the vari-able of nonviable sperm selection and were associated withreasonable fertilization rates in our patients.

References1. Rott HD. Kartagener’s syndrome and the syndrome of the immotile cilia.

Hum Genet 1979;46:249–61.2. Olmedo SB, Nodar F, Chillik C, Chemes HE. Successful intracytoplas-

mic sperm injection with spermatozoa from a patient with dysplasia ofthe fibrous sheath and chronic respiratory disease. Hum Reprod 1997;12:1497–9.

3. von Zumbusch A, Fiedler K, Mayerhofer A, Jebberger B, Ring J, VoghtHJ. Birth of healthy children after intracytoplasmic sperm injection intwo couples with male Kartagener’s syndrome. Fertil Steril 1998;70:643–6.

4. Kay VJ, Irvine DS. Successful in-vitro fertilization pregnancy withspermatozoa from a patient with Kartagener’s syndrome. Hum Reprod2000;15:135–8.

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