Patients with acephalic spermatozoa syndrome linked to SUN5 mutations have a favorable pregnancy outcome from ICSI

Patients with acephalic spermatozoa syndrome linked to SUN5 mutations have a favorable pregnancy... Abstract STUDY QUESTION Are Sad1 and UNC84 domain containing 5 (SUN5) mutations associated with the outcomes of ICSI in patients with acephalic spermatozoa syndrome (ASS)? SUMMARY ANSWER Despite highly abnormal sperm morphology, ASS patients with SUN5 mutations have a favorable pregnancy outcome following ICSI. WHAT IS KNOWN ALREADY ASS is a rare cause of infertility characterized by the production of a majority of headless spermatozoa, along with a small proportion of intact spermatozoa with an abnormal head–tail junction. Previous studies have demonstrated that SUN5 mutations may cause ASS. Several studies showed that ICSI could help patients with ASS father children. STUDY DESIGN, SIZE, DURATION This retrospective cohort study included 11 infertile ASS males with SUN5 mutations. Five of them underwent five ICSI cycles. Their ICSI results were compared to men with ASS without SUN5 mutations (n = 3) and to men with multiple morphological abnormalities of the sperm flagella (MMAF) (n = 9). All ICSI treatments were completed between Jan 2011 and May 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS Sanger DNA sequencing was used to detect mutations in SUN5. Clinical and biological data were collected from patients at the fertility center. MAIN RESULTS AND THE ROLE OF CHANCE Sanger sequencing validated 11 patients with SUN5 mutations. Three novel mutations in SUN5 (c.829C>T [p.Q277*]; c.1067G>A [p.R356H]; c.211+1 insGT [p.S71Cfs11*]) were identified in three patients. The rates of fertilization, good-quality embryos and pregnancy for five patients with SUN5 mutations following ICSI were 81.5%, 81.8% and 100%, respectively. The rates of fertilization and good-quality embryos in patients with MMAF were significantly lower compared with ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively). There were no differences in ICSI results between ASS patients with and without SUN5 mutations. LIMITATIONS, REASONS FOR CAUTION Only a small number patients with SUN5 mutations was available because of its rare incidence. WIDER IMPLICATIONS OF THE FINDINGS Patients with ASS can be effectively treated with ICSI. SUN5 mutations may be one of the genetic causes of ASS. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Natural Science Foundation of China (81401251, 81370754, and 81170559), the Jiangsu Province Special Program of Medical Science (BL2012009, ZX201110, FXK201221) and a project funded by PAPD of the Priority Academic Program Development of Jiangsu High Education Institutions (JX10231802). None of the authors have any competing interests. SUN5, mutation, teratozoospermia, acephalic spermatozoa syndrome, ICSI Introduction Acephalic spermatozoa syndrome (ASS), a rare type of teratozoospermia, has been reported, but its cause remains poorly understood (Perotti et al., 1981; Rondanino et al., 2015). The major spermatozoa anomaly in ASS patients is headless spermatozoa. These usually occur along with some intact spermatozoa with abnormal head–tail junctions. Electron microscopy can often detect the absence of the implantation fossa and basal plate between the sperm head and tail (Chemes et al., 1999). ASS was previously suggested as a genetic disease. It repeats identically in different patients, and has familial incidence in humans (Baccetti et al., 1989; Chemes et al., 1999). Our previous research found that eight out of 17 ASS patients (47.06%) had genetic defects in Sad1 and UNC84 domain containing 5 (SUN5) (Zhu et al., 2016). The concentration and motility of sperm in ASS patients are often low, but some motile spermatozoa with abnormal head–tail junctions could be found, implying a possibility of successful pregnancy through ICSI. Herein, we present the ICSI results from five ASS patients with SUN5 mutations. We also report three novel mutations of SUN5 from three newly diagnosed ASS patients. Materials and Methods Patients and controls During Jan 2011–May 2017, 11 ASS patients with SUN5 mutations attended our fertility center. Eight patients who had been reported previously (Zhu et al., 2016) and three new patients (P5, P6 and P7) were involved in this study. Five ASS patients (P1, P2, P3, P4 and P5) with SUN5 mutations had completed ICSI procedures. Four of them (P1, P2, P3 and P4) were from a previous study (Zhu et al., 2016) and one (P5) was only reported in this study (Table I). The remaining six patients have not had an ICSI procedure; four of them have selected IUI or IVF-embryo transfer with donor sperm. Testicular volume, sex hormone levels and chromosome karyotype were all within normal limits. The ICSI outcomes of SUN5 mutated and SUN5 non-mutated ASS and MMAF men were compared. The ethical committee of the First Affiliated Hospital of Nanjing Medical University has approved this study. We obtained written informed consents from each participant. Table I Main semen characteristics and SUN5 mutations in seven patients with acephalic spermatozoa syndrome. Variables  SUN5  Sperm parameters*  Percentage of different morphological types#  Patients  Mutation  Volume (ml)  concentration (106/ml)  PR (%)  Motility (%)  Normal (%)  Abnormal head–tail junction (%)  Decaudated (%)  Acephalic (%)  P1  c.824C>T/c.381delA  3.9  1.5  4.6  18.3  0  1.6  0.1  98.3  P2  c.216G>A/c. 1043A>T  3  3.7  6.2  8.6  0  4.4  0.4  95.2  P3  c.851C>G  1.8  6.5  5.4  20.2  0  4.1  0.5  95.4  P4  c.425+1G>A/c.1043A>T  1.2  7.6  1.2  3.6  0  0.9  0.3  98.8  P5  c.829C>T/c.1066C>T  2  3.8  9.5  15.8  0  2.1  1.4  96.4  P6  c.1067G>A  2.6  2.2  6.7  11.2  0  1.7  1.1  97.7  P7  c.425+1G>A/c.211+1 insGT  1.9  5.2  7.2  13.1  0  2.7  0.5  96.8  Variables  SUN5  Sperm parameters*  Percentage of different morphological types#  Patients  Mutation  Volume (ml)  concentration (106/ml)  PR (%)  Motility (%)  Normal (%)  Abnormal head–tail junction (%)  Decaudated (%)  Acephalic (%)  P1  c.824C>T/c.381delA  3.9  1.5  4.6  18.3  0  1.6  0.1  98.3  P2  c.216G>A/c. 1043A>T  3  3.7  6.2  8.6  0  4.4  0.4  95.2  P3  c.851C>G  1.8  6.5  5.4  20.2  0  4.1  0.5  95.4  P4  c.425+1G>A/c.1043A>T  1.2  7.6  1.2  3.6  0  0.9  0.3  98.8  P5  c.829C>T/c.1066C>T  2  3.8  9.5  15.8  0  2.1  1.4  96.4  P6  c.1067G>A  2.6  2.2  6.7  11.2  0  1.7  1.1  97.7  P7  c.425+1G>A/c.211+1 insGT  1.9  5.2  7.2  13.1  0  2.7  0.5  96.8  *Acephalic and decaudated spermatozoa were not counted as spermatozoa here. #Acephalic and decaudated spermatozoa were counted as spermatozoa here. SUN5: Sad1 and UNC84 domain containing 5; PR: progressive motility. Semen analysis and sperm morphology Semen volume, sperm concentration, morphology and motility were evaluated at least twice according to guideline (WHO, 2010). Sperm morphology was assessed using the Shorr method. The morphological abnormalities were classified into four categories: normal, abnormal head–tail junction, decaudated and acephalic. The percentages of each category were calculated. Sanger sequencing of SUN5 The SUN5 gene was sequenced as described previously (Zhu et al., 2016). PCR products for sequencing SUN5 and deletion mapping were amplified using Q5 Taq polymerase (New England Biolabs, MA, USA). Western blot analysis Western blot was carried out as described previously (Zhu et al., 2016). A rabbit anti-SUN5 antibody (Rabbit Polyclonal, Proteintech, 1:1000) and a rabbit anti-GAPDH antibody (Rabbit Polyclonal, Keygene Biotech, 1:1000) were used for the Western blot. ICSI, embryo culture and embryo transfer ICSI was carried out as previously described (Shen et al., 2013). Only motile spermatozoa with a head and tail, including the abnormal junction of head–tail, were chosen for ICSI. The inseminated oocyte was cultured in Quinn’s medium (Sage, CT, USA). Embryos were transferred using ultrasound guidance. Statistical analyses The χ2 test was used to compare the three groups, such as the two pronuclei (2PN) rate, good-quality embryo rate, and transferrable embryo rate. Student’s t-test was used to compare the means between groups. Data are presented as mean ± SD. P values ≤ 0.05 were considered as statistically significant difference. All statistical analyses were performed with R software (version3.4.1; The R Foundation for Statistical Computer, http://www.cran.r-project.org/). Results Sanger sequencing analysis on ASS patients Mutations in P1, P2, P3 and P4 have been previously reported (Zhu et al., 2016). Our sequencing analysis identified three new cases (P5, P6 and P7) with novel mutations of SUN5 gene (Table I). Namely, P5 carried a compound heterozygous SUN5 variant (c.1066C>T[p.R356C]/c.829C>T[p.Q277*]). P6 carried one homozygous variant (c.1067G>A[p.R356H]). Another compound heterozygous variant (c.425+1G>A/c.211+1 insGT[p.S71Cfs11*]) was identified in P7 (Table I and Fig. 1). DNA samples from the parents of P5, P6 and P7 were sequenced, and the variants were found to be inherited from them. The segregation of the variants was established by sequencing two parents of the patients. With the in silico tools, including the SIFT (sorting intolerant from tolerant), PolyPhen-2, MutationTaster and NNSplice, we evaluated the predicted effect of the three novel mutations on protein function. The three novel mutations (c.829C>T; c.829 c.1067G>A; c.211+1 insGT) were found to be pathogenic. Moreover, the three novel mutations had a low allele frequency (0-19/121,238) in the ExAC dataset, and none were in a homozygous state (Table II). Table II Effects of novel SUN5 mutations predicted using in silico tools. Chromosome 20 co-ordinatesa  cDNA alteration  Amino acid alteration  Exon  Mutation  ExAC allele frequency  ExAC homozygotes frequency  NNSplice  SIFT  PolyPhen  Mutation Taster  31571673C>T  c.1067G>A  p. R356H  13  Missense  1/120294  0/120294  –  0.00 (D)  0.999 (D)  1.000 (D)b  31573610G>A  c. 829C>T  p.Q277*  11  Nonsense  Not found  Not found  –  –  –  1.000 (D)  31590390T>TA   c.211+1 insGT  p.S71Cfs11*  3  Frame-shift  19/121238  0/121238  –  –  –  1.000 (D)c  Chromosome 20 co-ordinatesa  cDNA alteration  Amino acid alteration  Exon  Mutation  ExAC allele frequency  ExAC homozygotes frequency  NNSplice  SIFT  PolyPhen  Mutation Taster  31571673C>T  c.1067G>A  p. R356H  13  Missense  1/120294  0/120294  –  0.00 (D)  0.999 (D)  1.000 (D)b  31573610G>A  c. 829C>T  p.Q277*  11  Nonsense  Not found  Not found  –  –  –  1.000 (D)  31590390T>TA   c.211+1 insGT  p.S71Cfs11*  3  Frame-shift  19/121238  0/121238  –  –  –  1.000 (D)c  aAll data are based on GRCh37/hg19. bThe letter D in the brackets indicates deleterious. cThe mutation 211+1 insGT was predicted to cause frame shift. Figure 1 View largeDownload slide Pedigrees of the three families with SUN5 mutations. DNA samples from the parents of P5, P6 and P7 were sequenced, revealing that the variants in Sad1 and UNC84 domain containing 5 (SUN5) were inherited from their parents. The carrier status of the parents is shown in the pedigree. Figure 1 View largeDownload slide Pedigrees of the three families with SUN5 mutations. DNA samples from the parents of P5, P6 and P7 were sequenced, revealing that the variants in Sad1 and UNC84 domain containing 5 (SUN5) were inherited from their parents. The carrier status of the parents is shown in the pedigree. The novel mutation (c.829C>T) inherited from a heterozygous mother was predicted to be a loss-of-function variant. The novel missense mutation (c.1067G>A [p.R356H]) is similar to the known missense mutation (c.1066C>T[p.R356C]). That mutation disrupted highly conserved amino acids in the C-terminal SUN domain and interfered in the interaction of SUN5 with its partners in the perinuclear space (Zhu et al., 2016). In addition, the frame-shift variant (c.211+1 insGT) of P7 in the N-terminal nucleoplasmic region predictably led to a premature termination codon (PTC) in SUN5 mRNA, producing a truncated SUN5 protein without its C-terminal SUN domain (Table II). Analysis of the SUN5 levels in two ASS cases with SUN5 mutations We analyzed the protein levels of SUN5 by Western blot in the ejaculated semen from two patients, P5 & P6. The semen from a healthy donor was used as a positive control. As shown in Fig. 2C, SUN5 protein was nearly undetectable in semen samples from P5 and P6. Figure 2 View largeDownload slide Analysis of sperm from infertile men with acephalic spermatozoa syndrome. (A and B) Photo of ejaculated semen stained using the Shorr method. A. Morphology of normal sperm; B. Morphology of sperm from patients with acephalic spermatozoa syndrome (ASS): headless sperm (black arrow), sperm with abnormal head–tail junction (red arrow); C. Western blotting showing the protein level of SUN5 in ejaculated semen from one control (lane 1) and two affected individuals (lanes 2 and 3) with SUN5 mutations (P5 and P6). Figure 2 View largeDownload slide Analysis of sperm from infertile men with acephalic spermatozoa syndrome. (A and B) Photo of ejaculated semen stained using the Shorr method. A. Morphology of normal sperm; B. Morphology of sperm from patients with acephalic spermatozoa syndrome (ASS): headless sperm (black arrow), sperm with abnormal head–tail junction (red arrow); C. Western blotting showing the protein level of SUN5 in ejaculated semen from one control (lane 1) and two affected individuals (lanes 2 and 3) with SUN5 mutations (P5 and P6). ICSI outcomes and semen parameters Semen analysis of patients with ASS showed that ~95% spermatozoa were acephalic and a small percentage had abnormal head–tail junctions or heads without tails (Fig. 2A and B, Table I). All patients were oligozoospermic and asthenozoospermic (Table I). In total, five couples underwent five ICSI cycles and the mean number of metaphase II (MII) oocytes was 10.80 ± 1.43 (mean ± SD). The rates of fertilization 2PN/injected oocytes and good-quality embryos (good-quality embryo/2PN) were 81.5 and 81.8%, respectively. Embryos from all five couples were frozen to prevent the development of ovarian hyperstimulation syndrome. The partners of the five patients all became pregnant following the first frozen embryo transfer cycle (Table III). There was one twin pregnancy (for P1). Minor malformation of the left ear dysplasia with mild hearing loss was diagnosed in one child (for P2). Table III Outcomes of ICSI cycles in the five patients with ASS. Variables  P1  P2  P3  P4  P5  Male age (years)  27  33  30  26  22  Female age (years)  23  34  30  27  21  Metaphase II oocytes (n)  15  7  10  9  13  2PN(n)  12  7  8  4  13  Good-quality embryos (n)  10  7  8  4  7  Blastocyst (n)  /  /  6  2  6  Frozen-all  Yes  Yes  Yes  Yes  Yes  Transferred embryos (n)  2  2  1  1  1  Clinical pregnancy  Yes  Yes  Yes  Yes  Yes  Delivery (n)  Girl(2)  Boy(1)  Ongoing  Ongoing  Ongoing  Variables  P1  P2  P3  P4  P5  Male age (years)  27  33  30  26  22  Female age (years)  23  34  30  27  21  Metaphase II oocytes (n)  15  7  10  9  13  2PN(n)  12  7  8  4  13  Good-quality embryos (n)  10  7  8  4  7  Blastocyst (n)  /  /  6  2  6  Frozen-all  Yes  Yes  Yes  Yes  Yes  Transferred embryos (n)  2  2  1  1  1  Clinical pregnancy  Yes  Yes  Yes  Yes  Yes  Delivery (n)  Girl(2)  Boy(1)  Ongoing  Ongoing  Ongoing  ASS, acephalic spermatozoa syndrome; 2PN, two pronuclei. To compare ICSI results, we evaluated the ICSI outcomes of three ASS patients without SUN5 mutations. There were no statistically significant differences in ICSI results between patients with SUN5 mutations and patients without SUN5 mutations. We also assessed the ICSI outcomes of nine MMAF patients; four had been reported in our previous study (Yang et al., 2016). There were no significant differences between the three groups in terms of age, MII oocytes, transferrable embryos, implantation, and pregnancy, miscarriage (Table IV). However, the rate of fertilization and good-quality embryos in MMAF patients was significantly lower compared with the ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively) (Table IV). Table IV Outcomes of ICSI in the three patient groups for the first ICSI and first embryo transfer cycle. Variable  ASS SUN5+  ASS SUN5−  MMAF  Patients (n)  5  3  9  Mean female age (years)  27.00 ± 5.24  31.33 ± 11.06  29.00 ± 3.74  Mean male age (years)  27.60 ± 4.16  31.00 ± 9.54  30.56 ± 5.15  MII oocytes (n)  54  20  96  2PN (%)  44/54(81.5)  17/20(85.0)  63/96(65.6)*  Transferrable embryo (%)  41/44(93.2)  17/17(100)  49/56(87.5)  Good-quality embryo (%)  36/44(81.8)  16/17(94.1)  30/56(53.6)*  Transferred embryos (n)  1.4 ± 0.55  2 ± 0  1.56 ± 0.53  Implantation (%)  85.7  50.0  35.7  Clinical pregnancy (%)  5/5(100)  3/3(100)  4/9(44.4)  Miscarriage (%)  0/5(0)  0/3(0)  1/4(25.0)  Variable  ASS SUN5+  ASS SUN5−  MMAF  Patients (n)  5  3  9  Mean female age (years)  27.00 ± 5.24  31.33 ± 11.06  29.00 ± 3.74  Mean male age (years)  27.60 ± 4.16  31.00 ± 9.54  30.56 ± 5.15  MII oocytes (n)  54  20  96  2PN (%)  44/54(81.5)  17/20(85.0)  63/96(65.6)*  Transferrable embryo (%)  41/44(93.2)  17/17(100)  49/56(87.5)  Good-quality embryo (%)  36/44(81.8)  16/17(94.1)  30/56(53.6)*  Transferred embryos (n)  1.4 ± 0.55  2 ± 0  1.56 ± 0.53  Implantation (%)  85.7  50.0  35.7  Clinical pregnancy (%)  5/5(100)  3/3(100)  4/9(44.4)  Miscarriage (%)  0/5(0)  0/3(0)  1/4(25.0)  * P ≤ 0.05 for MAFF versus SUN5+ and SUN5. The χ2 test was used to compare the proportions between three groups (e.g. of 2PN rate, good-quality embryo rate Student’s t-test was used to compare the means). Data are mean ± SD. MMAF, multiple morphological abnormalities of the sperm flagella. Discussion Teratozoospermia, such as MMAF, globozoospermia and ASS, has been reported as a cause of male infertility. ASS is rarely found in infertile males. Light microscopy can detect a large number of progressive motile headless tails, and a few spermatozoa with an abnormal angle between the head and tail. Our previous work reported that biallelic SUN5 mutations may cause autosomal-recessive ASS, and one recent report confirmed the homozygous deletion of SUN5 in patients with ASS from Algeria (Elkhatib et al., 2017). Herein, our new Sanger sequencing analysis of the SUN5 gene identified three novel mutations. Western blotting analysis using samples from P5 and P6 demonstrated that the SUN5 mutations reduced protein levels, similar to the finding in P3 and P4 (Zhu et al., 2016). These results indicat that the reduced SUN5 protein level after gene mutation may be responsible, at least in part, for the development of ASS. Despite ASS being a serious teratozoospermia, ICSI can help patients to father children if they have intact motile spermatozoa, even if the head–tail junction is abnormal. Porcu et al. (2003) reported a successful pregnancy from ICSI using decapitated sperm from two brothers. However, an earlier study reported a case of pregnancy failure after ICSI with decapitated sperm, despite having good-quality embryos (Saias-Magnan et al., 1999). So far, for all five ASS patients with SUN5 mutations who had ICSI, fertilization was achieved. Therefore, the abnormal head–tail junction may not affect the fertilization rate and clinical pregnancy. In addition, we also achieved a pregnancy rate of 100% in three ASS patients without SUN5 mutations using ICSI; however, the number was rather limited. This indicates that ICSI may be an optimal therapy for ASS patients with motile spermatozoa but with an abnormal head–tail junction. Pregnancy following ICSI in MMAF patients has been previously described (Yang et al., 2016). The rate of transferable embryos and pregnancy was no difference between the three groups in the present study. The rate of fertilization and good-quality embryos in MMAF patients was significantly lower compared with ASS patients (Table IV). Two previous studies showed that patients with tail pathologies had a good prognosis following ICSI (Chemes and Rawe, 2003; Wambergue et al., 2016). The discrepancy is probably because our MMAF patients had no, or lower frequency, dynein axonemal heavy chain 1 (DNAH1) mutations. In addition, a few of our MMAF patients did not have enough motile spermatozoa to perform ICSI. Nevertheless, the number of our patients was rather limited and further studies are needed to validate the pathological effects of the abnormal head–tail junction and tail of spermatozoa on the outcome of ICSI. In summary, we have identified three novel mutations of the SUN5 gene that possibly lead to ASS. ICSI is an optimal therapy with a favorable pregnancy outcome. Nevertheless, further studies are needed to define the precise molecular mechanisms of how SUN5 mutations lead to ASS. Authors’ roles F.J. undertook the molecular work and handled the recruitment of patients, sample collection and sperm analysis. Z.J. and C.Y. analyzed the data and wrote the manuscript and designed the study. Y.X., Z.F. and L.J. supervised all molecular laboratory work, had full access to all the data in the study and take responsibility for the integrity of the data and its accuracy. All authors contributed to the report. Funding This study was supported by The National Natural Science Foundation of China (81401251, 81370754, 81170559), the Jiangsu Province Special Program of Medical Science (BL2012009, ZX201110, FXK201221) and a project funded by PAPD of the Priority Academic Program Development of Jiangsu High Education Institutions (JX10231802). Conflict of interest None declared. References Baccetti B, Burrini AG, Collodel G, Magnano AR, Piomboni P, Renieri T, Sensini C. Morphogenesis of the decapitated and decaudated sperm defect in two brothers. Gamete Res  1989; 23: 181– 188. Google Scholar CrossRef Search ADS PubMed  Chemes EH, Puigdomenech ET, Carizza C, Olmedo SB, Zanchetti F, Hermes R. Acephalic spermatozoa and abnormal development of the head–neck attachment: a human syndrome of genetic origin. Hum Reprod  1999; 14: 1811– 1818. Google Scholar CrossRef Search ADS PubMed  Chemes EH, Rawe YV. Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men. Hum Reprod Update  2003; 9: 405– 428. Google Scholar CrossRef Search ADS PubMed  Elkhatib RA, Paci M, Longepied G, Saias-Magnan J, Courbiere B, Guichaoua MR, Levy N, Metzler-Guillemain C, Mitchell MJ. Homozygous deletion of SUN5 in three men with decapitated spermatozoa. Hum Mol Genet  2017; 26: 3167– 3171. Google Scholar PubMed  Perotti ME, Giarola A, Gioria M. Ultrastructural study of the decapitated sperm defect in an infertile man. J Reprod Fertil  1981; 63: 543– 549. Google Scholar CrossRef Search ADS PubMed  Porcu G, Mercier G, Boyer P, Achard V, Banet J, Vasserot M, Melone C, Saias-Magnan J, D’Ercole C, Chau C et al.  . Pregnancies after ICSI using sperm with abnormal head–tail junction from two brothers: case report. Hum Reprod  2003; 18: 562– 567. Google Scholar CrossRef Search ADS PubMed  Rondanino C, Duchesne V, Escalier D, Jumeau F, Verhaeghe F, Peers MC, Mitchell V, Rives N. Evaluation of sperm nuclear integrity in patients with different percentages of decapitated sperm in ejaculates. Reprod Biomed Online  2015; 31: 89– 99. Google Scholar CrossRef Search ADS PubMed  Saias-Magnan J, Metzler-Guillemain C, Mercier G, Carles-Marcorelles F, Grillo JM, Guichaoua MR. Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa: case report. Hum Reprod  1999; 14: 1989– 1992. Google Scholar CrossRef Search ADS PubMed  Shen JD, Cram DS, Wu W, Cai LB, Yang XY, Sun XP, Cui YG, Liu JY. Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis. Reprod Biomed Online  2013; 27: 176– 183. Google Scholar CrossRef Search ADS PubMed  Wambergue C, Zouari R, Fourati Ben Mustapha S, Martinez G, Devillard F, Hennebicq S, Satre V, Brouillet S, Halouani L, Marrakchi O et al.  . Patients with multiple morphological abnormalities of the sperm flagella due to DNAH1 mutations have a good prognosis following intracytoplasmic sperm injection. Hum Reprod  2016; 31: 1164– 1172. Google Scholar CrossRef Search ADS PubMed  World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen , 5th edn. Cambridge: Cambridge University Press, 2010, 37– 44, 65–67. Yang SM, Yang XY, Ding Y, Li H, Wang W, Liu JY, Wen DG. Intracytoplasmic sperm injection outcomes in Chinese men with multiple morphological abnormalities of sperm flagella. Asian J Androl  2016; 18: 809– 811. Google Scholar CrossRef Search ADS PubMed  Zhu F, Wang F, Yang X, Zhang J, Wu H, Zhang Z, Zhang Z, He X, Zhou P, Wei Z et al.  . Biallelic SUN5 Mutations Cause Autosomal-Recessive acephalic spermatozoa syndrome. Am J Hum Genet  2016; 99: 942– 949. Google Scholar CrossRef Search ADS PubMed  Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Patients with acephalic spermatozoa syndrome linked to SUN5 mutations have a favorable pregnancy outcome from ICSI

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Oxford University Press
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Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2018.
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Abstract

Abstract STUDY QUESTION Are Sad1 and UNC84 domain containing 5 (SUN5) mutations associated with the outcomes of ICSI in patients with acephalic spermatozoa syndrome (ASS)? SUMMARY ANSWER Despite highly abnormal sperm morphology, ASS patients with SUN5 mutations have a favorable pregnancy outcome following ICSI. WHAT IS KNOWN ALREADY ASS is a rare cause of infertility characterized by the production of a majority of headless spermatozoa, along with a small proportion of intact spermatozoa with an abnormal head–tail junction. Previous studies have demonstrated that SUN5 mutations may cause ASS. Several studies showed that ICSI could help patients with ASS father children. STUDY DESIGN, SIZE, DURATION This retrospective cohort study included 11 infertile ASS males with SUN5 mutations. Five of them underwent five ICSI cycles. Their ICSI results were compared to men with ASS without SUN5 mutations (n = 3) and to men with multiple morphological abnormalities of the sperm flagella (MMAF) (n = 9). All ICSI treatments were completed between Jan 2011 and May 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS Sanger DNA sequencing was used to detect mutations in SUN5. Clinical and biological data were collected from patients at the fertility center. MAIN RESULTS AND THE ROLE OF CHANCE Sanger sequencing validated 11 patients with SUN5 mutations. Three novel mutations in SUN5 (c.829C>T [p.Q277*]; c.1067G>A [p.R356H]; c.211+1 insGT [p.S71Cfs11*]) were identified in three patients. The rates of fertilization, good-quality embryos and pregnancy for five patients with SUN5 mutations following ICSI were 81.5%, 81.8% and 100%, respectively. The rates of fertilization and good-quality embryos in patients with MMAF were significantly lower compared with ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively). There were no differences in ICSI results between ASS patients with and without SUN5 mutations. LIMITATIONS, REASONS FOR CAUTION Only a small number patients with SUN5 mutations was available because of its rare incidence. WIDER IMPLICATIONS OF THE FINDINGS Patients with ASS can be effectively treated with ICSI. SUN5 mutations may be one of the genetic causes of ASS. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Natural Science Foundation of China (81401251, 81370754, and 81170559), the Jiangsu Province Special Program of Medical Science (BL2012009, ZX201110, FXK201221) and a project funded by PAPD of the Priority Academic Program Development of Jiangsu High Education Institutions (JX10231802). None of the authors have any competing interests. SUN5, mutation, teratozoospermia, acephalic spermatozoa syndrome, ICSI Introduction Acephalic spermatozoa syndrome (ASS), a rare type of teratozoospermia, has been reported, but its cause remains poorly understood (Perotti et al., 1981; Rondanino et al., 2015). The major spermatozoa anomaly in ASS patients is headless spermatozoa. These usually occur along with some intact spermatozoa with abnormal head–tail junctions. Electron microscopy can often detect the absence of the implantation fossa and basal plate between the sperm head and tail (Chemes et al., 1999). ASS was previously suggested as a genetic disease. It repeats identically in different patients, and has familial incidence in humans (Baccetti et al., 1989; Chemes et al., 1999). Our previous research found that eight out of 17 ASS patients (47.06%) had genetic defects in Sad1 and UNC84 domain containing 5 (SUN5) (Zhu et al., 2016). The concentration and motility of sperm in ASS patients are often low, but some motile spermatozoa with abnormal head–tail junctions could be found, implying a possibility of successful pregnancy through ICSI. Herein, we present the ICSI results from five ASS patients with SUN5 mutations. We also report three novel mutations of SUN5 from three newly diagnosed ASS patients. Materials and Methods Patients and controls During Jan 2011–May 2017, 11 ASS patients with SUN5 mutations attended our fertility center. Eight patients who had been reported previously (Zhu et al., 2016) and three new patients (P5, P6 and P7) were involved in this study. Five ASS patients (P1, P2, P3, P4 and P5) with SUN5 mutations had completed ICSI procedures. Four of them (P1, P2, P3 and P4) were from a previous study (Zhu et al., 2016) and one (P5) was only reported in this study (Table I). The remaining six patients have not had an ICSI procedure; four of them have selected IUI or IVF-embryo transfer with donor sperm. Testicular volume, sex hormone levels and chromosome karyotype were all within normal limits. The ICSI outcomes of SUN5 mutated and SUN5 non-mutated ASS and MMAF men were compared. The ethical committee of the First Affiliated Hospital of Nanjing Medical University has approved this study. We obtained written informed consents from each participant. Table I Main semen characteristics and SUN5 mutations in seven patients with acephalic spermatozoa syndrome. Variables  SUN5  Sperm parameters*  Percentage of different morphological types#  Patients  Mutation  Volume (ml)  concentration (106/ml)  PR (%)  Motility (%)  Normal (%)  Abnormal head–tail junction (%)  Decaudated (%)  Acephalic (%)  P1  c.824C>T/c.381delA  3.9  1.5  4.6  18.3  0  1.6  0.1  98.3  P2  c.216G>A/c. 1043A>T  3  3.7  6.2  8.6  0  4.4  0.4  95.2  P3  c.851C>G  1.8  6.5  5.4  20.2  0  4.1  0.5  95.4  P4  c.425+1G>A/c.1043A>T  1.2  7.6  1.2  3.6  0  0.9  0.3  98.8  P5  c.829C>T/c.1066C>T  2  3.8  9.5  15.8  0  2.1  1.4  96.4  P6  c.1067G>A  2.6  2.2  6.7  11.2  0  1.7  1.1  97.7  P7  c.425+1G>A/c.211+1 insGT  1.9  5.2  7.2  13.1  0  2.7  0.5  96.8  Variables  SUN5  Sperm parameters*  Percentage of different morphological types#  Patients  Mutation  Volume (ml)  concentration (106/ml)  PR (%)  Motility (%)  Normal (%)  Abnormal head–tail junction (%)  Decaudated (%)  Acephalic (%)  P1  c.824C>T/c.381delA  3.9  1.5  4.6  18.3  0  1.6  0.1  98.3  P2  c.216G>A/c. 1043A>T  3  3.7  6.2  8.6  0  4.4  0.4  95.2  P3  c.851C>G  1.8  6.5  5.4  20.2  0  4.1  0.5  95.4  P4  c.425+1G>A/c.1043A>T  1.2  7.6  1.2  3.6  0  0.9  0.3  98.8  P5  c.829C>T/c.1066C>T  2  3.8  9.5  15.8  0  2.1  1.4  96.4  P6  c.1067G>A  2.6  2.2  6.7  11.2  0  1.7  1.1  97.7  P7  c.425+1G>A/c.211+1 insGT  1.9  5.2  7.2  13.1  0  2.7  0.5  96.8  *Acephalic and decaudated spermatozoa were not counted as spermatozoa here. #Acephalic and decaudated spermatozoa were counted as spermatozoa here. SUN5: Sad1 and UNC84 domain containing 5; PR: progressive motility. Semen analysis and sperm morphology Semen volume, sperm concentration, morphology and motility were evaluated at least twice according to guideline (WHO, 2010). Sperm morphology was assessed using the Shorr method. The morphological abnormalities were classified into four categories: normal, abnormal head–tail junction, decaudated and acephalic. The percentages of each category were calculated. Sanger sequencing of SUN5 The SUN5 gene was sequenced as described previously (Zhu et al., 2016). PCR products for sequencing SUN5 and deletion mapping were amplified using Q5 Taq polymerase (New England Biolabs, MA, USA). Western blot analysis Western blot was carried out as described previously (Zhu et al., 2016). A rabbit anti-SUN5 antibody (Rabbit Polyclonal, Proteintech, 1:1000) and a rabbit anti-GAPDH antibody (Rabbit Polyclonal, Keygene Biotech, 1:1000) were used for the Western blot. ICSI, embryo culture and embryo transfer ICSI was carried out as previously described (Shen et al., 2013). Only motile spermatozoa with a head and tail, including the abnormal junction of head–tail, were chosen for ICSI. The inseminated oocyte was cultured in Quinn’s medium (Sage, CT, USA). Embryos were transferred using ultrasound guidance. Statistical analyses The χ2 test was used to compare the three groups, such as the two pronuclei (2PN) rate, good-quality embryo rate, and transferrable embryo rate. Student’s t-test was used to compare the means between groups. Data are presented as mean ± SD. P values ≤ 0.05 were considered as statistically significant difference. All statistical analyses were performed with R software (version3.4.1; The R Foundation for Statistical Computer, http://www.cran.r-project.org/). Results Sanger sequencing analysis on ASS patients Mutations in P1, P2, P3 and P4 have been previously reported (Zhu et al., 2016). Our sequencing analysis identified three new cases (P5, P6 and P7) with novel mutations of SUN5 gene (Table I). Namely, P5 carried a compound heterozygous SUN5 variant (c.1066C>T[p.R356C]/c.829C>T[p.Q277*]). P6 carried one homozygous variant (c.1067G>A[p.R356H]). Another compound heterozygous variant (c.425+1G>A/c.211+1 insGT[p.S71Cfs11*]) was identified in P7 (Table I and Fig. 1). DNA samples from the parents of P5, P6 and P7 were sequenced, and the variants were found to be inherited from them. The segregation of the variants was established by sequencing two parents of the patients. With the in silico tools, including the SIFT (sorting intolerant from tolerant), PolyPhen-2, MutationTaster and NNSplice, we evaluated the predicted effect of the three novel mutations on protein function. The three novel mutations (c.829C>T; c.829 c.1067G>A; c.211+1 insGT) were found to be pathogenic. Moreover, the three novel mutations had a low allele frequency (0-19/121,238) in the ExAC dataset, and none were in a homozygous state (Table II). Table II Effects of novel SUN5 mutations predicted using in silico tools. Chromosome 20 co-ordinatesa  cDNA alteration  Amino acid alteration  Exon  Mutation  ExAC allele frequency  ExAC homozygotes frequency  NNSplice  SIFT  PolyPhen  Mutation Taster  31571673C>T  c.1067G>A  p. R356H  13  Missense  1/120294  0/120294  –  0.00 (D)  0.999 (D)  1.000 (D)b  31573610G>A  c. 829C>T  p.Q277*  11  Nonsense  Not found  Not found  –  –  –  1.000 (D)  31590390T>TA   c.211+1 insGT  p.S71Cfs11*  3  Frame-shift  19/121238  0/121238  –  –  –  1.000 (D)c  Chromosome 20 co-ordinatesa  cDNA alteration  Amino acid alteration  Exon  Mutation  ExAC allele frequency  ExAC homozygotes frequency  NNSplice  SIFT  PolyPhen  Mutation Taster  31571673C>T  c.1067G>A  p. R356H  13  Missense  1/120294  0/120294  –  0.00 (D)  0.999 (D)  1.000 (D)b  31573610G>A  c. 829C>T  p.Q277*  11  Nonsense  Not found  Not found  –  –  –  1.000 (D)  31590390T>TA   c.211+1 insGT  p.S71Cfs11*  3  Frame-shift  19/121238  0/121238  –  –  –  1.000 (D)c  aAll data are based on GRCh37/hg19. bThe letter D in the brackets indicates deleterious. cThe mutation 211+1 insGT was predicted to cause frame shift. Figure 1 View largeDownload slide Pedigrees of the three families with SUN5 mutations. DNA samples from the parents of P5, P6 and P7 were sequenced, revealing that the variants in Sad1 and UNC84 domain containing 5 (SUN5) were inherited from their parents. The carrier status of the parents is shown in the pedigree. Figure 1 View largeDownload slide Pedigrees of the three families with SUN5 mutations. DNA samples from the parents of P5, P6 and P7 were sequenced, revealing that the variants in Sad1 and UNC84 domain containing 5 (SUN5) were inherited from their parents. The carrier status of the parents is shown in the pedigree. The novel mutation (c.829C>T) inherited from a heterozygous mother was predicted to be a loss-of-function variant. The novel missense mutation (c.1067G>A [p.R356H]) is similar to the known missense mutation (c.1066C>T[p.R356C]). That mutation disrupted highly conserved amino acids in the C-terminal SUN domain and interfered in the interaction of SUN5 with its partners in the perinuclear space (Zhu et al., 2016). In addition, the frame-shift variant (c.211+1 insGT) of P7 in the N-terminal nucleoplasmic region predictably led to a premature termination codon (PTC) in SUN5 mRNA, producing a truncated SUN5 protein without its C-terminal SUN domain (Table II). Analysis of the SUN5 levels in two ASS cases with SUN5 mutations We analyzed the protein levels of SUN5 by Western blot in the ejaculated semen from two patients, P5 & P6. The semen from a healthy donor was used as a positive control. As shown in Fig. 2C, SUN5 protein was nearly undetectable in semen samples from P5 and P6. Figure 2 View largeDownload slide Analysis of sperm from infertile men with acephalic spermatozoa syndrome. (A and B) Photo of ejaculated semen stained using the Shorr method. A. Morphology of normal sperm; B. Morphology of sperm from patients with acephalic spermatozoa syndrome (ASS): headless sperm (black arrow), sperm with abnormal head–tail junction (red arrow); C. Western blotting showing the protein level of SUN5 in ejaculated semen from one control (lane 1) and two affected individuals (lanes 2 and 3) with SUN5 mutations (P5 and P6). Figure 2 View largeDownload slide Analysis of sperm from infertile men with acephalic spermatozoa syndrome. (A and B) Photo of ejaculated semen stained using the Shorr method. A. Morphology of normal sperm; B. Morphology of sperm from patients with acephalic spermatozoa syndrome (ASS): headless sperm (black arrow), sperm with abnormal head–tail junction (red arrow); C. Western blotting showing the protein level of SUN5 in ejaculated semen from one control (lane 1) and two affected individuals (lanes 2 and 3) with SUN5 mutations (P5 and P6). ICSI outcomes and semen parameters Semen analysis of patients with ASS showed that ~95% spermatozoa were acephalic and a small percentage had abnormal head–tail junctions or heads without tails (Fig. 2A and B, Table I). All patients were oligozoospermic and asthenozoospermic (Table I). In total, five couples underwent five ICSI cycles and the mean number of metaphase II (MII) oocytes was 10.80 ± 1.43 (mean ± SD). The rates of fertilization 2PN/injected oocytes and good-quality embryos (good-quality embryo/2PN) were 81.5 and 81.8%, respectively. Embryos from all five couples were frozen to prevent the development of ovarian hyperstimulation syndrome. The partners of the five patients all became pregnant following the first frozen embryo transfer cycle (Table III). There was one twin pregnancy (for P1). Minor malformation of the left ear dysplasia with mild hearing loss was diagnosed in one child (for P2). Table III Outcomes of ICSI cycles in the five patients with ASS. Variables  P1  P2  P3  P4  P5  Male age (years)  27  33  30  26  22  Female age (years)  23  34  30  27  21  Metaphase II oocytes (n)  15  7  10  9  13  2PN(n)  12  7  8  4  13  Good-quality embryos (n)  10  7  8  4  7  Blastocyst (n)  /  /  6  2  6  Frozen-all  Yes  Yes  Yes  Yes  Yes  Transferred embryos (n)  2  2  1  1  1  Clinical pregnancy  Yes  Yes  Yes  Yes  Yes  Delivery (n)  Girl(2)  Boy(1)  Ongoing  Ongoing  Ongoing  Variables  P1  P2  P3  P4  P5  Male age (years)  27  33  30  26  22  Female age (years)  23  34  30  27  21  Metaphase II oocytes (n)  15  7  10  9  13  2PN(n)  12  7  8  4  13  Good-quality embryos (n)  10  7  8  4  7  Blastocyst (n)  /  /  6  2  6  Frozen-all  Yes  Yes  Yes  Yes  Yes  Transferred embryos (n)  2  2  1  1  1  Clinical pregnancy  Yes  Yes  Yes  Yes  Yes  Delivery (n)  Girl(2)  Boy(1)  Ongoing  Ongoing  Ongoing  ASS, acephalic spermatozoa syndrome; 2PN, two pronuclei. To compare ICSI results, we evaluated the ICSI outcomes of three ASS patients without SUN5 mutations. There were no statistically significant differences in ICSI results between patients with SUN5 mutations and patients without SUN5 mutations. We also assessed the ICSI outcomes of nine MMAF patients; four had been reported in our previous study (Yang et al., 2016). There were no significant differences between the three groups in terms of age, MII oocytes, transferrable embryos, implantation, and pregnancy, miscarriage (Table IV). However, the rate of fertilization and good-quality embryos in MMAF patients was significantly lower compared with the ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively) (Table IV). Table IV Outcomes of ICSI in the three patient groups for the first ICSI and first embryo transfer cycle. Variable  ASS SUN5+  ASS SUN5−  MMAF  Patients (n)  5  3  9  Mean female age (years)  27.00 ± 5.24  31.33 ± 11.06  29.00 ± 3.74  Mean male age (years)  27.60 ± 4.16  31.00 ± 9.54  30.56 ± 5.15  MII oocytes (n)  54  20  96  2PN (%)  44/54(81.5)  17/20(85.0)  63/96(65.6)*  Transferrable embryo (%)  41/44(93.2)  17/17(100)  49/56(87.5)  Good-quality embryo (%)  36/44(81.8)  16/17(94.1)  30/56(53.6)*  Transferred embryos (n)  1.4 ± 0.55  2 ± 0  1.56 ± 0.53  Implantation (%)  85.7  50.0  35.7  Clinical pregnancy (%)  5/5(100)  3/3(100)  4/9(44.4)  Miscarriage (%)  0/5(0)  0/3(0)  1/4(25.0)  Variable  ASS SUN5+  ASS SUN5−  MMAF  Patients (n)  5  3  9  Mean female age (years)  27.00 ± 5.24  31.33 ± 11.06  29.00 ± 3.74  Mean male age (years)  27.60 ± 4.16  31.00 ± 9.54  30.56 ± 5.15  MII oocytes (n)  54  20  96  2PN (%)  44/54(81.5)  17/20(85.0)  63/96(65.6)*  Transferrable embryo (%)  41/44(93.2)  17/17(100)  49/56(87.5)  Good-quality embryo (%)  36/44(81.8)  16/17(94.1)  30/56(53.6)*  Transferred embryos (n)  1.4 ± 0.55  2 ± 0  1.56 ± 0.53  Implantation (%)  85.7  50.0  35.7  Clinical pregnancy (%)  5/5(100)  3/3(100)  4/9(44.4)  Miscarriage (%)  0/5(0)  0/3(0)  1/4(25.0)  * P ≤ 0.05 for MAFF versus SUN5+ and SUN5. The χ2 test was used to compare the proportions between three groups (e.g. of 2PN rate, good-quality embryo rate Student’s t-test was used to compare the means). Data are mean ± SD. MMAF, multiple morphological abnormalities of the sperm flagella. Discussion Teratozoospermia, such as MMAF, globozoospermia and ASS, has been reported as a cause of male infertility. ASS is rarely found in infertile males. Light microscopy can detect a large number of progressive motile headless tails, and a few spermatozoa with an abnormal angle between the head and tail. Our previous work reported that biallelic SUN5 mutations may cause autosomal-recessive ASS, and one recent report confirmed the homozygous deletion of SUN5 in patients with ASS from Algeria (Elkhatib et al., 2017). Herein, our new Sanger sequencing analysis of the SUN5 gene identified three novel mutations. Western blotting analysis using samples from P5 and P6 demonstrated that the SUN5 mutations reduced protein levels, similar to the finding in P3 and P4 (Zhu et al., 2016). These results indicat that the reduced SUN5 protein level after gene mutation may be responsible, at least in part, for the development of ASS. Despite ASS being a serious teratozoospermia, ICSI can help patients to father children if they have intact motile spermatozoa, even if the head–tail junction is abnormal. Porcu et al. (2003) reported a successful pregnancy from ICSI using decapitated sperm from two brothers. However, an earlier study reported a case of pregnancy failure after ICSI with decapitated sperm, despite having good-quality embryos (Saias-Magnan et al., 1999). So far, for all five ASS patients with SUN5 mutations who had ICSI, fertilization was achieved. Therefore, the abnormal head–tail junction may not affect the fertilization rate and clinical pregnancy. In addition, we also achieved a pregnancy rate of 100% in three ASS patients without SUN5 mutations using ICSI; however, the number was rather limited. This indicates that ICSI may be an optimal therapy for ASS patients with motile spermatozoa but with an abnormal head–tail junction. Pregnancy following ICSI in MMAF patients has been previously described (Yang et al., 2016). The rate of transferable embryos and pregnancy was no difference between the three groups in the present study. The rate of fertilization and good-quality embryos in MMAF patients was significantly lower compared with ASS patients (Table IV). Two previous studies showed that patients with tail pathologies had a good prognosis following ICSI (Chemes and Rawe, 2003; Wambergue et al., 2016). The discrepancy is probably because our MMAF patients had no, or lower frequency, dynein axonemal heavy chain 1 (DNAH1) mutations. In addition, a few of our MMAF patients did not have enough motile spermatozoa to perform ICSI. Nevertheless, the number of our patients was rather limited and further studies are needed to validate the pathological effects of the abnormal head–tail junction and tail of spermatozoa on the outcome of ICSI. In summary, we have identified three novel mutations of the SUN5 gene that possibly lead to ASS. ICSI is an optimal therapy with a favorable pregnancy outcome. Nevertheless, further studies are needed to define the precise molecular mechanisms of how SUN5 mutations lead to ASS. Authors’ roles F.J. undertook the molecular work and handled the recruitment of patients, sample collection and sperm analysis. Z.J. and C.Y. analyzed the data and wrote the manuscript and designed the study. Y.X., Z.F. and L.J. supervised all molecular laboratory work, had full access to all the data in the study and take responsibility for the integrity of the data and its accuracy. All authors contributed to the report. Funding This study was supported by The National Natural Science Foundation of China (81401251, 81370754, 81170559), the Jiangsu Province Special Program of Medical Science (BL2012009, ZX201110, FXK201221) and a project funded by PAPD of the Priority Academic Program Development of Jiangsu High Education Institutions (JX10231802). Conflict of interest None declared. References Baccetti B, Burrini AG, Collodel G, Magnano AR, Piomboni P, Renieri T, Sensini C. Morphogenesis of the decapitated and decaudated sperm defect in two brothers. Gamete Res  1989; 23: 181– 188. Google Scholar CrossRef Search ADS PubMed  Chemes EH, Puigdomenech ET, Carizza C, Olmedo SB, Zanchetti F, Hermes R. Acephalic spermatozoa and abnormal development of the head–neck attachment: a human syndrome of genetic origin. Hum Reprod  1999; 14: 1811– 1818. Google Scholar CrossRef Search ADS PubMed  Chemes EH, Rawe YV. Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men. Hum Reprod Update  2003; 9: 405– 428. Google Scholar CrossRef Search ADS PubMed  Elkhatib RA, Paci M, Longepied G, Saias-Magnan J, Courbiere B, Guichaoua MR, Levy N, Metzler-Guillemain C, Mitchell MJ. Homozygous deletion of SUN5 in three men with decapitated spermatozoa. Hum Mol Genet  2017; 26: 3167– 3171. Google Scholar PubMed  Perotti ME, Giarola A, Gioria M. Ultrastructural study of the decapitated sperm defect in an infertile man. J Reprod Fertil  1981; 63: 543– 549. Google Scholar CrossRef Search ADS PubMed  Porcu G, Mercier G, Boyer P, Achard V, Banet J, Vasserot M, Melone C, Saias-Magnan J, D’Ercole C, Chau C et al.  . Pregnancies after ICSI using sperm with abnormal head–tail junction from two brothers: case report. Hum Reprod  2003; 18: 562– 567. Google Scholar CrossRef Search ADS PubMed  Rondanino C, Duchesne V, Escalier D, Jumeau F, Verhaeghe F, Peers MC, Mitchell V, Rives N. Evaluation of sperm nuclear integrity in patients with different percentages of decapitated sperm in ejaculates. Reprod Biomed Online  2015; 31: 89– 99. Google Scholar CrossRef Search ADS PubMed  Saias-Magnan J, Metzler-Guillemain C, Mercier G, Carles-Marcorelles F, Grillo JM, Guichaoua MR. Failure of pregnancy after intracytoplasmic sperm injection with decapitated spermatozoa: case report. Hum Reprod  1999; 14: 1989– 1992. Google Scholar CrossRef Search ADS PubMed  Shen JD, Cram DS, Wu W, Cai LB, Yang XY, Sun XP, Cui YG, Liu JY. Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis. Reprod Biomed Online  2013; 27: 176– 183. Google Scholar CrossRef Search ADS PubMed  Wambergue C, Zouari R, Fourati Ben Mustapha S, Martinez G, Devillard F, Hennebicq S, Satre V, Brouillet S, Halouani L, Marrakchi O et al.  . Patients with multiple morphological abnormalities of the sperm flagella due to DNAH1 mutations have a good prognosis following intracytoplasmic sperm injection. Hum Reprod  2016; 31: 1164– 1172. Google Scholar CrossRef Search ADS PubMed  World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen , 5th edn. Cambridge: Cambridge University Press, 2010, 37– 44, 65–67. Yang SM, Yang XY, Ding Y, Li H, Wang W, Liu JY, Wen DG. Intracytoplasmic sperm injection outcomes in Chinese men with multiple morphological abnormalities of sperm flagella. Asian J Androl  2016; 18: 809– 811. Google Scholar CrossRef Search ADS PubMed  Zhu F, Wang F, Yang X, Zhang J, Wu H, Zhang Z, Zhang Z, He X, Zhou P, Wei Z et al.  . Biallelic SUN5 Mutations Cause Autosomal-Recessive acephalic spermatozoa syndrome. Am J Hum Genet  2016; 99: 942– 949. Google Scholar CrossRef Search ADS PubMed  Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Human ReproductionOxford University Press

Published: Mar 1, 2018

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