CASE REPORT Can intracytoplasmic morphologically selected sperm injection be used to select normal-sized sperm heads in infertile patients with macrocephalic sperm head syndrome? Mohamed Hassen Chelli, Pharm.D., a Martine Albert, M.D., a,b Pierre F. Ray, Ph.D., c Bruno Guthauser, Pharm.D., d Vincent Izard, M.D., e Ibrahim Hammoud, M.D., a,b Jacqueline Selva, Ph.D., a,b and Francxois Vialard, Ph.D. a,b a Department of Reproductive Biology and Cytogenetics, Centre Hospitalier Poissy, Saint Germain, France; b EA 2493, Universite Versailles, St. Quentin en Yvelines, France; c Department of Genetics and Procreation, Centre Hospitalier Universitaire de Grenoble, La Tronche, France; d Department of Reproductive Biology, Centre Hospitalier, Dreux, France; and e Department of Urology, Centre Hospitalier de Bic^etre, Le Kremlin Bic^etre, France Objective: To study the chromosomal content of selected by intracytoplasmic morphologically selected sperm injection (IMSI) in cases of macrocephalic sperm head syndrome. Design: Case report. Setting: Obstetrics, gynecology, urology, and reproductive biology departments. Patient(s): Two infertile patients with large-headed. Intervention(s): Fluorescence in situ hybridization on selected with normal-sized heads after IMSI selection. Main Outcome Measure(s): Percentages of polyploid, diploid, haploid aneuploid, and normal. Result(s): Of the six that could be selected, all were haploid but aneuploid. Conclusion(s): Absence of normal haploid among high magnification selected contraindicated IMSI for these two patients. (Fertil Steril Ò 2010;93:1347.e1 e5. Ó2010 by American Society for Reproductive Medicine.) Key Words: FISH, large-headed, normal-head-size, high magnification, IMSI, macrocephalic sperm head syndrome, meiotic division deficiency Received August 25, 2008; revised October 1, 2008; accepted October 29, 2008; published online December 4, 2008. M.H.C. has nothing to disclose. M.A. has nothing to disclose. P.F.R. has nothing to disclose. B.G. has nothing to disclose. V.I. has nothing to disclose. I.H. has nothing to disclose. J.S. has nothing to disclose. J.V. has nothing to disclose. Reprint requests: Francxois Vialard, Ph.D., Department of Reproductive Biology, Cytogenetics, Gynecology and Obstetrics, CHI Poissy Saint Germain, 10 rue du champs Gaillard, 78303 Poissy, France (FAX: 33-1-39-27-44-25; E-mail: fvialard@hotmail.com). Intracytoplasmic sperm injection (ICSI) has increased the chances of procreation for patients with spermatogenesis deficiency (1). This assisted reproductive technique is widely used in male patients with compromised sperm quality, such as severe teratozoospermia (2), asthenozoospermia (3), or oligozoospermia (4, 5). Intracytoplasmic sperm injection, however, seems inefficient in cases of macrocephalic sperm head syndrome, which is strongly associated with abnormal sperm chromosomal content, aneuploidy, and/or polyploidy (2). Recently it has been suggested that sperm fluorescence in situ hybridization (FISH) and genetic counseling should be done for men with more than 20% macrocephalic sperm (6). This syndrome was first described in 1977 (7) and is associated with large and abnormally shaped heads and multiple flagella. Recently a homozygous mutation in the aurora kinase C gene (AURKC) (8) was described as a genetic cause of large-headed multiflagellar polyploid sperm production. AURKC is a cell cycle regulatory serine/threonine kinase found mainly in the testis, and its expression seems to be restricted to meiosis. Normal-head in this type of sperm syndrome can be retrieved through lengthy and careful microscopic examination, but we have shown that these are frequently aneuploid (9). We thus concluded that normal sperm morphology at ICSI magnification does not guarantee normal chromosomal content and is not sufficient to indicate ICSI in cases of macrocephalic sperm head syndrome (9). Bartoov et al. (10, 11) have developed a high-magnification observation technique allowing better evaluation of motile sperm morphology, known as motile sperm organelle morphology examination (MSOME). Sperm selection is carried out at 6,000 magnification by this technique, allowing better 0015-0282/10/$36.00 Fertility and Sterility â Vol. 93, No. 4, March 1, 2010 1347.e1 doi:10.1016/j.fertnstert.2008.10.059 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
sperm nucleus assessment. Adapted to injection, it takes the name intracytoplasmic morphologically selected sperm injection (IMSI). The first results published suggest that IMSI allows higher implantation and pregnancy rates, especially after two ICSI failures (12). We thus wanted to evaluate whether the morphologic selection of by high magnification would permit identification of euploid in cases of macrocephalic sperm head syndrome. We present here the results of FISH analysis carried out on evaluated as being suitable for injection after [1] ICSI selection (normal-sized heads in ICSI), and [2] MSOME selection according to Bartoov s normalcy morphologic criteria by high-magnification screening (11). These analyses were done for two infertile men with macrocephalic sperm head syndrome, to determine whether ICSI or IMSI could be applied successfully to these patients. CASE REPORT The two infertile patients with macrocephalic sperm head syndrome originated from North Africa. According to World Health Organization criteria the patients were asthenozoospermic, with no progressive motility and 10% slow motility associated with normal sperm count (98.7 10 6 /ml) for patient 1 and no progressive motility and 16% slow motility associated with normal sperm count (42.5 10 6 /ml) for patient 2. Total teratozoospermia (100%) was found for both patients, and sperm analysis showed 96% macrocephalic forms for patient 1 and 94% for patient 2. The two patients had no history of orchitis, toxic exposure, trauma, testicular torsion, or undescended testicle. There was no febrile episode in the months before semen analysis. Testicular volume was normal for both patients. Follicle-stimulating hormone levels were, respectively, 2.8 IU/L and 1.9 IU/L for patients 1 and 2. Karyotype was normal for both. After genetic counseling, each patient provided informed consent for sperm chromosomal evaluation and genetic analysis. These cases belong to a protocol dealing with genetic aspects of infertility approved by our local ethics committee and reported to the French health authorities. Molecular Biology AURKC sequence analysis was carried out for both patients. Genomic DNA was extracted from peripheral blood leukocytes using a guanidinium chloride extraction procedure. Sequence analysis was carried out using the BigDye Terminator v3.1 sequencing kit and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Primers and protocol are as reported by Dieterich et al. (8). Sequencing of both strands of AURKC exon 3 was carried out for each patient. whole sperm, and one for sperm preparation and subsequent microscopic examination for morphologic selection. Whole Sperm FISH Analysis After seminal liquid elimination, whole sperm was washed twice with sterile water (300 g for 10 minutes) and fixed by Carnoy acetic solution. Spermatozoa were then spread on a slide for FISH study. Sperm Preparation for ICSI and IMSI Migration of was performed on PureSperm density gradient (PureSperm 100 Nidacon; JCD, La Mulatiere, France) (45% 90%) 300 g for 20 minutes and sperm pellet washed with Universal IVF Medium (MediCult, Limonset, France) (600 g for 10 minutes). Previous reports showed that the selection of morphologically normal was time consuming in this syndrome. We thus set ourselves a time limit to try to select 100 by both ICSI and IMSI (MSOME) (3 hours for each procedure for each patient). Selection for ICSI was done under an inverted microscope (Diaphot, 400 objective; Nikon, Tokyo, Japan), and with normal head size were individually aspirated in the ICSI pipette (Humagen, Charlottesville, VA; inner diameter, 7 mm). Selection by MSOME was done under an inverted microscope and Nomarsky contrast (TU 2000 E, 1,000 objective; Nikon), and corresponding to Bartoov s definition (11) were individually aspirated in the same ICSI pipette. Selected were dropped and grouped on a microscopic slide. The FISH analysis was performed on those selected. FISH on Selected Spermatozoa Both selection slides were air dried and fixed with methanol for 5 minutes at room temperature, and decondensation was performed in a 3N NaOH solution (1 minute). After dehydration, FISH was performed with X, Y, 18 centromeric probes (Abbott Laboratories, Chicago, IL) with a 73 C 4-mn, 37 C overnight hybridization program. Slides were washed and counterstained with 6-diamino-2-phenylindole solution, and were analyzed with a Zeiss microscope (Carl Zeiss, Jena, Germany) using Pathvision Software (Digital Scientific, Cambridge, United Kingdom). Observation and interpretation criteria were based on the number of spots for X, Y, and 18 chromosomes on the sperm nuclei. Statistical analysis was performed using the Statview program c 2 test (Adept Scientific, Bethesda, MD). Differences were considered significant at P<.05. Semen Preparation and FISH Analysis For each patient, the same sperm sample was divided into two parts after seminal liquefaction: one for FISH analysis on RESULTS Sequence analysis showed that both patients were homozygous for the AURKC c.144delc deletion. 1347.e2 Chelli et al. IMSI in macrocephalic sperm head syndrome Vol. 93, No. 4, March 1, 2010
TABLE 1 Sperm chromosomal analysis according to selection method. Patient 1 Patient 2 MSOME-selected ICSI-selected Unselected MSOME-selected ICSI-selected Unselected Parameter 1,012 31 6 1,002 8 0 No. of analyzed Polyploid, n (%) 788 (77.8) 0 0 870 (86.8) 2 (25.0) Diploid, n (%) 200 (19.8) 16 (51.6) 0 103 (10.3) 1 (12.5) Haploid aneuploid, 14 (1.4) 15 (48.4) 6 (100.0) 26 (2.6) 4 (50.0) n (%) 10 (1.0) 1 (3.2) 0 3 (0.3) 1 (12.5) Haploid euploid (normal), n (%) Chelli. IMSI in macrocephalic sperm head syndrome. Fertil Steril 2010. For patient 1, 1,012 unselected were analyzed. Only 10 (1.0%) were scored as normal for the three analyzed chromosomes; 14 (1.4%) were haploid but aneuploid, 200 (19.8%) diploid, and 788 (77.8%) polyploid. Among the 31 ICSI-selected, only 1 (3.2%) was diagnosed as normal; 15 (48.4%) were haploid but aneuploid and 16 (51.6%) diploid. All of the 6 MSOME-selected were haploid but aneuploid. For patient 2, 1,002 unselected were analyzed. Only 3 (0.3%) were scored as normal for the three analyzed chromosomes; 26 (2.6%) were haploid but aneuploid, 103 (10.3%) diploid, and 870 (86.8%) polyploid. Among the 8 ICSI-selected, only 1 (12.5%) was diagnosed as normal; 4 (50.0%) were haploid but aneuploid, 1 (12.5%) was diploid, and 2 (25.0%) were polyploid. No MSOME-selected were found for this patient. For the two patients, we were not able to select 100 with either ICSI or IMSI. In total, only 39 were ICSI selected and 6 IMSI selected. Global results are summarized in Table 1 and Figure 1. For the two patients, we observed a statistically significant (P<.0001) decrease in polyploidy and an increase in diploidy and haploidy between whole sperm and ICSI-selected. We also observed a statistically significant (P<.05) decrease in diploidy and an increase in haploidy between ICSI-selected and MSOME-selected. DISCUSSION The molecular diagnosis confirmed that both our patients carried the recurrent c.144delc deletion leading to classic large-headed. Our objective was to evaluate the utility of IMSI in infertile patients with macrocephalic sperm head syndrome male infertility, and its ability to select euploid. For each patient, with the same sperm sample, we compared aneuploidy rates by FISH in three situations: for whole sperm, after ICSI selection, and after MSOME selection. Generally, because of low aneuploidy rates, sperm FISH was performed on a minimum of 1 to 10,000 (13). To avoid misinterpreting FISH results because of the low number of analyzed (maximum 100 per patient and selection), we estimate that the following conclusions can be drawn because the whole-sperm aneuploidy rate is estimated to be up to 99% and nearly 90% after ICSI selection (9) in this syndrome. Other syndromes with major teratozoospermia (14) or asthenospermia (4) in patients with a normal karyotype are associated with increased aneuploidy risk, but the reported levels of chromosomal abnormalities were never greater than 5%. As expected, we experienced difficulties in increasing the number of of normal size and morphology. Very few could be selected: 39 by ICSI, 6 by MSOME. These results were in accordance with our previous Fertility and Sterility â 1347.e3
FIGURE 1 Spermatozoa distribution according to selection and chromosomal analysis. Chelli. IMSI in macrocephalic sperm head syndrome. Fertil Steril 2010. report, whereby only 79 were retrieved after 5 hours of ICSI selection (9). Sperm aneuploidy rates in whole semen were nearly 99%, as expected, and were comparable to those of previous studies (15 18), showing essentially diploidy and polyploidy. In these two patients we confirmed previous results (9), which indicated that morphologic selection of performed during ICSI (head size allowing ICSI pipette aspiration) is not efficient to predict normal chromosomal content. Only 2 of 39 ICSI-selected were normal (6 of 79 in the previous report) (9). Most were diploid or haploid aneuploid. In contrast, all MSOME-selected were haploid, and none were diploid or polyploid. Selection by MSOME resulted in statistically significant elimination of sperm polyploidy and diploidy (MSOME vs. whole sperm, P<.0001; MSOME vs. ICSI, P<.05) but without selection of euploid. It thus seems that, with high magnification, we were able to eliminate the head distension likely associated with diploidy or polyploidy. These results should be viewed with caution because only 6 were retrieved, but they may reflect both the efficacy and the limits of MSOME selection. In this study, although MSOME selection eliminated polyploid, it could not identify euploid because all the selected were aneuploid. This result confirms that selection of with normal head shape by ICSI or IMSI in macrocephalic sperm head syndrome is not sufficient to guarantee normal chromosomal content. Chromosomal risk for these patients remains high, probably near 100% if we test all chromosomes. We can extrapolate from these results and speculate that MSOME cannot detect aneuploid haploid but can help to eliminate more severe chromosomal defects, such as polyploidy. Improvement in clinical outcomes with IMSI is probably due to its ability to detect such growth chromosomal defects, perhaps as well as more subtle chromatin abnormalities or DNA damage. To our knowledge, the present study provides the first report of a FISH analysis specifically performed on MSOMEselected for patients with macrocephalic sperm head syndrome. However, we found fewer polyploid sperm in ICSI selection than in our previous report (9), perhaps suggesting that MSOME influenced us in our ICSI selection technique and prompted us to pay more attention to sperm morphology. Finally, for the two patients, only aneuploid haploid MSOME-selected were found. The high risk of conceiving abnormal offspring (polyploidy and aneuploid embryo), and a likely unsuccessful outcome, led us to inform the two patients that IMSI, like ICSI, was a contraindicated procedures. Sperm donation programs and adoption were discussed. In conclusion, we confirmed that the presence of normalhead-size is not a sufficient condition to indicate ICSI in cases of macrocephalic sperm head syndrome associated with recurrent AURKC c.144delc (9). On the other hand, no normal haploid were retrieved by high-magnification selection. Conversely, it seems that we were able to select haploid with MSOME, suggesting that the larger, more aberrant had the most abnormal (polyploid) chromosomal content. Diploid and polyploid probably have distended heads. The MSOMEselected were, however, exceptional: only six were retrieved after a 6-hour selection procedure, and all were aneuploid. This suggests that the improvement of sperm ploidy after MSOME is not due to a better selection of sperm chromosomal quality but probably to a better sperm nuclear structure. Finally, the absence of normal haploid among high magnification selected sperm formally contraindicated IMSI for our two patients and most probably for all AURKC c.144delc homozygous patients. REFERENCES 1. Faure AK, Aknin-Seifer I, Frerot G, Pelletier R, De Robertis C, Cans C, et al. Predictive factors for an increased risk of sperm aneuploidies in oligo-astheno-teratozoospermic males. Int J Androl 2007;30:153 62. 2. Viville S, Mollard R, Bach ML, Falquet C, Gerlinger P, Warter S. Do morphological anomalies reflect chromosomal aneuploidies? Case report. Hum Reprod 2000;15:2563 6. 3. Tesarik J, Mendoza C. Treatment of severe male infertility by micromanipulation-assisted fertilization: an update. Front Biosci 2007;12:105 14. 4. Rives N, Mousset-Simeon N, Mazurier S, Mace B. 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