Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin

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1 Reproductive BioMedicine Online (2010) 20, ARTICLE Sperm nuclear vacuoles, as assessed by motile sperm organellar morphological examination, are mostly of acrosomal origin O Kacem a, C Sifer b, V Barraud-Lange a, B Ducot c, D De Ziegler d, C Poirot e, JP Wolf a, * a Service d Histologie-Embryologie-Biologie de la Reproduction, Hôpital Cochin, AP-HP, 123, Bd Port Royal Paris, Université Paris Descartes, France; b Service d Histologie-Embryologie-Cytogénétique, Hôpital Jean Verdier, AP-HP, Avenue du 14 Juillet, Bondy, France; c Inserm, U822 Epidémiologie, Démographie et Sciences Sociales, IFR69, INED, Le Kremlin-Bicêtre, France; d Service de Gynécologie-Obstétrique 2, UF de Médecine de la Reproduction, Hôpital Cochin, AP-HP, Université Paris Descartes, 123, Bd Port Royal Paris, Université Paris Descartes, France; e Unité Fonctionnelle de Biologie de la Reproduction, Hôpital Pitié Salpêtrière, Université Pierre et Marie Curie (UPMC), Bd de l Hôpital, Paris, France * Corresponding author. address: jean-philippe.wolf@cch.aphp.fr (JP Wolf). Olfa Kacem received her MD from the Faculty of Medicine, University of Tunis, with specialization in cytogenetics and the biology of reproduction. From she was a member of the Histology-Embryology- Cytogenetics Department, Jean Verdier Hospital, AP-HP, France. In 2007, she received her MMed in the Biology of Reproduction (Paris V). Currently she is Assistant Professor in the Department of Medicine and Biology of Reproduction, Aziza Othmana Hospital, Tunis. Abstract Microinjection of nuclear vacuole-free spermatozoa selected by motile sperm organellar morphological examination (MSOME) has been claimed to enhance assisted reproduction treatment outcome compared with intracytoplasmic sperm injection. However, the nature of these nuclear vacuoles is unclear, since their localization at the front of the sperm head suggests they might be of acrosomal origin. To study this hypothesis, acrosomal status was evaluated using Pisum sativum agglutinin staining on a smear, together with sperm organellar morphological examination using the same optics as for MSOME on 30 sperm samples from infertile patients, yielding >3200 spermatozoa. Vacuoles were present in 61% of spermatozoa when acrosomal material or intact acrosomes were observed, versus 29% when spermatozoa were acrosome reacted (P < ). Induction of the acrosomal reaction by ionophore A23587 from 17.4% to 36.1% significantly increased the percentage of vacuole-free spermatozoa from 41.2% to 63.8% (P < 0.001). These data suggest that most nuclear vacuoles are of acrosomal origin. Hence, the best spermatozoa selected by MSOME are mostly acrosome-reacted spermatozoa. As microinjection of spermatozoa with a persistent acrosome drastically hampers embryo development in animal models, this suggests that the improvement in pregnancy rates reported following intracytoplasmic injection of morphologically selected sperm might be due to the procedure allowing injection of acrosome-reacted spermatozoa. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: acrosome reaction, Nomarski optics, sperm nuclear vacuoles /$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 Sperm nuclear vacuoles and acrosomal status 133 Introduction Recently, a new technique of sperm selection prior to intracytoplasmic sperm injection (ICSI) has been developed (intracytoplasmic morphologically selected sperm injection; IMSI), which has been claimed to enhance pregnancy and lower miscarriage rates when compared with ICSI (Berkovitz et al., 2006a,b). IMSI consists of the intracytoplasmic injection of a spermatozoon that has been selected based on motile sperm organellar morphological examination (MSOME) (Bartoov et al., 2001). MSOME is an unstained real-time high magnification motile sperm examination performed using an inverted microscope equipped with interferential contrast Normarski optics. Maximal optical magnification ( 100), magnification selector ( 1.5) and video-coupled magnification lead to a final video magnification around Criteria for normally shaped nuclei are smooth, symmetric, oval configuration and homogeneity of the nuclear chromatin mass ( nuclear vacuoles not exceeding more than 4% of the nuclear area). It is not clear where the nuclear vacuoles that are seen at this magnification on the sperm head are located. Only transmission electron microscopy would accurately locate them (Zamboni, 1987), but they have been described by Bartoov et al. (2002) as chromatin vacuoles. Although the improvement of clinical outcome with IMSI compared with ICSI has not yet been clearly established by a prospective randomized study, the nature of the so-called nuclear vacuoles as assessed by MSOME has been questioned. Indeed chromatin vacuoles are unlikely to be detected using differential interference contrast (DIC) microscopy at a magnification of 6600 for three reasons. First, for chromatin vacuoles, to be seen, electron microscopy examination with magnification around 20,000 or more (Zamboni, 1987) is required. Second, DIC only allows images of surfaces by reflection of birefringent light rays and does not allow analysis of intracellular structures (Hoffman and Gross, 1968, 1975; Padawer, 1968). Third, these vacuoles are mostly located in the anterior part of the sperm head, where the acrosome is located. This suggests that these images may be more likely linked to the presence of the acrosome or of acrosomal material rather than to a chromatin defect. To study this hypothesis, the presence of acrosome was evaluated using Pisum sativum agglutinin (PSA) staining along with sperm organellar morphology examination on sperm smear preparations. Materials and methods Thirty patients consulting at the infertility clinic of the Jean Verdier hospital (Assistance Publique, Hôpitaux de Paris) were selected. Mean sperm parameters were: concentration: 65.6 ± /ml ( /ml), progressive motility 43 ± 18% (10 65%), vitality: 79 ± 7.8% (45 94%), normal morphology according to David s criteria 29 ± 4.6% (5 65%) (Jouannet et al., 1981). Progressive motility of selected spermatozoa was 74 ± 0.5% (30 95%). Most patients had >80% motile spermatozoa (only one patient had 30%). A total of 3237 spermatozoa were studied corresponding to an average number of 100 spermatozoa per patient. Sperm organellar morphological examination and assessment of the acrosomal status Sperm organellar morphological examination and assessment of the acrosomal status were simultaneously performed on the same smear, on immotile spermatozoa. Spermatozoa were allowed to liquefy for 30 min and selection of motile spermatozoa was performed using a twoconcentration PurespermÒ gradient (95% and 45%). The pellet was then washed and diluted in Ferticult HEPES to a final concentration of spermatozoa/ml. Sperm smears were done on SuperFrost slides and fixed with 4% paraformaldehyde for 30 min at room temperature. When dried, acrosome staining was performed with PSA-FITC (fluorescein isothiocyanate) 1/200 in a humidified chamber for 15 min (Mendoza et al., 1992). Slides were then washed twice in phospate-buffered saline ( 1) and with distilled water and mounted with Vectashield/4(,6- diamidino-2-phenylindole) under a coverslip. If slides were not read immediately, they were kept in the dark at 4 C. Immunofluorescence and sperm organellar morphological examination were both analysed on fixed spermatozoa using an inverted Nikon TE2000-S microscope equipped with a 60 immersion objective with Nomarski contrast according to the technique described by Bartoov et al. (2001, 2002, 2003) for MSOME. After sperm morphology had been analysed, the acrosome status of the same spermatozoon was studied on the same microscope using epifluorescence. Image analysis was done using a LUCIA G image analyser (Nikon, Japan) on a HP Workstation xw4100. Merging the two pictures representing the morphology and immunofluorescence of the same immotile spermatozoon was achieved using Adobe Photoshop package ( Sperm samples from 10 patients were also analysed for their acrosomal status and morphology using the same technique of smear analysis, before and after the acrosome reaction had been induced by ionophore A23587, 10 mmol/l. Motile sperm organellar morphological examination (MSOME) Analysis of motile spermatozoa was also performed according to the MSOME technique of Bartoov (2001, 2002, 2003). Movies were recorded using the LUCIA G image analyser (Nikon) on a HP Workstation and isolated consecutive images of the same spermatozoon were analysed. Statistical analysis Distribution of the 3237 spermatozoa into two groups: acrosome reacted (group 1), incomplete acrosome reaction or intact acrosome (group 2), according to the presence of nuclear vacuoles, was analysed using the chi-squared test with stratification on patient. The presence of vacuoles before and after induction of acrosome reaction in 10 patients was compared by Wilcoxon matched-pairs signed-ranks test. Differences were considered statistically significant for P < 0.05.

3 134 O Kacem et al. Results Description of nuclear vacuoles and of acrosomal status at magnification 6600 Immotile spermatozoa were analysed by the sperm organellar morphological examination technique and then immunofluorescence for their acrosomal status on the same smear. Normal spermatozoa with a complete acrosome reaction showed a regular shape of the head with no or very few vacuoles (Figure 1A and B). When the same spermatozoa were analysed for acrosomal status, they showed no or very little green fluorescence in the area of the head (Figure 1A 0 and B 0 ), indicating their status as acrosome-reacted spermatozoa. Interestingly, analysis by immunofluorescence of the acrosomal status of spermatozoa that presented large nuclear vacuoles (Figure 2A and B) showed green staining in the anterior part of the sperm head corresponding to the acrosome location, and in most cases an increase of brightness in the region corresponding to the vacuoles (arrow, Figure 2A 0 and B 0 ). This correspondence, presented on the merge photo (Figure 2A 00 and B 00 ), suggests that the image of the vacuole is of acrosomal origin. This was the case for spermatozoa eliciting an incomplete acrosome reaction (Figure 2A) and those for which the acrosome was considered intact (Figure 2B). Presence of nuclear vacuoles and the acrosomal status Table 1 shows the distribution of nuclear vacuoles according to the acrosome status (acrosome reacted group 1) or acrosome intact, or incomplete acrosome reaction (group 2) for 30 infertile patients. As shown, the percentage of spontaneous acrosomal reacted spermatozoa was 20.9%. Twenty-nine percent of acrosome-reacted spermatozoa presented vacuoles, while this percentage rose to 60.7% of spermatozoa with intact or incomplete acrosome reaction (P < ). Among 1750 sperm with vacuoles, 88.7% had an intact acrosome or acrosomal material. It is concluded that most vacuoles detected by MSOME are of acrosomal origin. This suggests that first class spermatozoa, defined as having no nuclear vacuole by MSOME analysis, are in fact mostly spermatozoa that have undergone a complete acrosome reaction. Induction of the acrosome reaction significantly reduces the percentage of vacuolated spermatozoa Should the nuclear vacuoles be linked to the presence of some acrosomal material, induction of the acrosome reaction would increase the percentage of vacuole-free spermatozoa corresponding to an increase of the percentage of spermatozoa with a complete acrosome reaction. This was in fact the case. Sperm samples from 10 patients were analysed for their acrosomal status and organellar morphological examination, before and after induction of the acrosome reaction by ionophore A23587, 10 mmol/l for 30 min. In selected spermatozoa, the mean percentage of spontaneous acrosome-reacted spermatozoa was 17.4% (Table 2) and the percentage of vacuole-free spermatozoa 41.2%. Induction of the acrosome reaction by ionophore A23587 resulted in an increase of the mean percentage of vacuole-free spermatozoa from 41.2% to 63.8% P < 0.005, whereas the percentage of acrosome-reacted spermatozoa increased from to (P < 0.001). This finding reinforces the link between vacuoles as assessed by MSOME and the presence of acrosomal material. MSOME analysis of acrosome reacting spermatozoa Motile acrosome reacting spermatozoa were analysed by MSOME. Large protruding blebs could be seen on live motile spermatozoa suggesting an ongoing acrosome reaction Figure 1 Acrosome detection on normal spermatozoa, according to sperm organellar morphological examination. Spermatozoa were immobilized and dried on a smear. The smear was then analysed by Normaski interferential contrast (A and B) and for the acrosomal status using the Pisum sativum agglutinin fluorescein isothiocyanate (PSA-FITC) detection technique and immunofluorescence (A 0 and B 0 ). Merge (A 00 and B 00 ). Note the absence of fluorescence after PSA-FITC staining ( 6600).

4 Sperm nuclear vacuoles and acrosomal status 135 Figure 2 Acrosome detection on spermatozoa with nuclear vacuoles, according to sperm organellar morphological examination. Spermatozoa with nuclear vacuoles (see Figure 1) ( 6600) (A) spermatozoon with incomplete acrosome reaction, (B) acrosome intact. Immunofluorescence (A 0 and B 0 ). Merge (A 00 and B 00 ). Highlighted green spots can be seen in the location of the vacuoles (arrows). These spermatozoa had a fluorescent zone on all the anterior head suggesting an intact acrosome or incomplete acrosome reaction ( 6600). Table 1 Presence of nucleur vacuoles in sperm heads according to the sperm acrosomal status. Vacuoles Absent Present Total Acrosome-reacted sperm 481 (70.9) 197 (29.1) 678 (20.9) Incomplete acrosome reaction or 1006 (39.3) 1553 (60.7) 2559 (79.1) intact acrosome Total Values are n or n (%). Comparison of the set of data using chi-squared analysis revealed a highly statistically significantly different distribution of vacuoles according to whether the spermatozoa were acrosome reacted or not (P < ). Table 2 Percentage of spermatozoa without nuclear vacuoles according to acrosomal status before and after induction of the acrosome reaction. Patient Spontaneous Induced Acrosome reacted Vacuole-free spermatozoa Acrosome reacted Vacuole-free spermatozoa Mean ± SD 17.4 ± ± ± 12.7 a 63.8 ± 11.0 b a Significantly different from spontaneous acrosome reaction, P < b Significantly different from percentage of vacuole-free spermatozoa in untreated group, P <

5 136 O Kacem et al. (Figure 3A and B, pictures are taken 2 seconds apart). When seen upfront, these blebs may appear as large vacuoles. The next spermatozoon analysed by MSOME on two consecutive pictures shows an embossed image on Figure 3C and an image of a vacuole in the same place on Figure 3D. Discussion This study provides evidence that the presence of nuclear vacuoles on sperm heads, as assessed by MSOME, is mostly associated with the presence of acrosomal material. However, one should explain why immunofluorescence staining of the acrosome shows highlighted spots at the place of vacuoles, where a fluorescence defect would be expected. There are several explanations. (i) As already stated, the DIC allows only images of surfaces by reflection of birefringent light rays and does not allow analysis of intracellular structures (Hoffman and Gross, 1968, 1975; Padawer, 1968). It does not show the difference between a depression and an outgrowth and should be interpreted with caution. In fact, as shown in Figure 3C and D, the same spermatozoon analysed by MSOME in two consecutive pictures shows an embossed image on Figure 3C and an image of a vacuole at the same place on Figure 3D. This means that what is interpreted as a vacuole and a defect of material may in fact be the presence of excessive material. (ii) Analysis of sperm structures by electron microscopy frequently shows acrosomal abnormalities. As reported by Zamboni (1987), acrosome malformations may appear as herniae within the sperm nucleus, including the presence of some acrosomal material and membranes within the nucleus. This material would be stained by PSA and therefore appear as highlighted spots. All these reasons, which are not mutually exclusive, can explain the presence of acrosomal material where a vacuole is seen, and can therefore explain the presence of highlighted spots. However, of greatest importance is that all these images are present when there is positive acrosomal staining, suggesting a link between nuclear vacuoles seen by MSOME and the presence of acrosome material. Interestingly, theses data suggest that the vacuole-free spermatozoa that are microinjected during IMSI are mostly acrosome-reacted spermatozoa. This hypothesis is further supported by two sets of data. Firstly, a large set of experimental data from animal models have demonstrated a toxic effect of acrosomal content on embryo development and pup delivery (Roldan, 2006). In the hamster, injection of an acrosome intact spermatozoon results in the death of the oocyte and removal of the acrosome is mandatory prior to microinjection in order to achieve successful ICSI (Yamauchi et al., 2002). In mice, the treatment of spermatozoa with calcium ionophore to induce the acrosome reaction before ICSI increases acrosome-free spermatozoa from 28% to 58%, and pronuclear formation to approximately 60% (range 59 62% across experiments) in intact oocytes (Lacham-Kaplan and Trounson, 1995). Always in the mouse, if the removal of sperm plasma membrane and acrosome is not a prerequisite to produce offspring by ICSI, it results in earlier onset of oocyte activation and better embryonic development with an increase of live born pups from 40.2% to 71.5% of transferred embryos (Morozumi et al., 2006). The components of the acrosome harmful to the oocyte are likely to be acrosomal enzymes such as trypsin-like acrosin and hyaluronidase because injection of commercially available trypsin and hyaluronidase mimicked the effects of injected supernumerary acrosome-intact spermatozoa (Morozumi and Yanagimachi, 2005). Secondly, in humans the ratio of the acrosome to ooplasm volume is much smaller than in animal models. Furthermore, immobilization of spermatozoa for ICSI by compressing and rolling the sperm tails induces variable disruption and sometimes loss of the acrosome (Gomez-Torres et al., 2007). This could be a reason for the higher success rates when ICSI is performed using immobilized spermatozoa (Takeuchi et al., 2004). However, the developmental potential of an Figure 3 Live motile spermatozoa analysed by motile sperm organellar morphological examination at consecutive time points. The same spermatozoon with a nuclear vacuole is shown on two consecutive pictures. Panels (A) and (B) show a live motile spermatozoon with protruding blebs (arrows) in the acrosomal region. It could be interpreted as an ongoing acrosome reaction. A different spermatozoon in shown panel (C) exhibits an embossed image. However, the same image looks like a defect of material on the same spermatozoon in (D) (arrows) ( 6600).

6 Sperm nuclear vacuoles and acrosomal status 137 ICSI embryo is not as good as that of an IVF embryo. Indeed, the percentage of blastocyst formation is significantly smaller in ICSI embryos (Griffiths et al., 2000). Furthermore, recently, Gianaroli et al. (2008) demonstrated in humans that microinjection of acrosome-free spermatozoa improved ICSI clinical outcome and delivery rate per attempt. The implantation rate rose from 8.6% for oocytes injected with non-reacted spermatozoa, to 39.0% for those injected with reacted spermatozoa. The delivery rate per cycle followed the same trend, while there was no effect on the fertilizing capacity and embryo development of either type of spermatozoon (Gianaroli et al., 2008). In a recent study, Peer et al. reported a significant decrease in the morphological integrity of sperm nuclei after 2 h of incubation at 37 C (Peer et al., 2007). They claimed that the high incidence of early spontaneous abortion post-icsi may be linked to this sperm defect. But another hypothesis could be that these vacuoles result from an increasing percentage of ongoing acrosome reaction similar to that reported in this study (Figure 3A). On the other hand, the absence of the acrosome on the microinjected spermatozoa in IMSI may not fully account for the improved results. Indeed, the normal shape of the head as defined by MSOME may exclude some genetically abnormal spermatozoa. Examination of human sperm chromosomes after sperm injection into mouse oocytes revealed that spermatozoa with abnormal head morphology have a significantly higher incidence of chromosome abnormality than those with normal heads, although they are not necessarily genomically abnormal (Yanagimachi, 1998). This may prevent some early pregnancy termination. Altogether, these data strongly support the hypothesis that human embryo development and pregnancy rate following ICSI may be improved by microinjection of acrosome-reacted spermatozoa. References Bartoov, B., Berkovitz, A., Eltes, F., Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil. Steril. 80, Bartoov, B., Berkovitz, A., Eltes, F., et al., Real-time fine morphology of motile human sperm cells is associated with IVF ICSI outcome. J. Androl. 23, 1 8. Bartoov, B., Berkovitz, A., Eltes, F., Selection of spermatozoa with normal nuclei to improve the pregnancy rate with intracytoplasmic sperm injection. N. Engl. J. Med. 345, Berkovitz, A., Eltes, F., Lederman, H., et al., 2006a. How to improve IVF ICSI outcome by sperm selection. Reprod. BioMed. Online 12, Berkovitz, A., Eltes, F., Ellenbogen, A., et al., 2006b. 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Steril. 48, Declaration: The authors report no financial or commercial conflicts of interest. Received 6 March 2009; refereed 22 April 2009; accepted 16 September 2009.