Received: 16 July 2019 | Revised: 17 September 2019 | Accepted: 21 September 2019 DOI: 10.1111/and.13459

INVITED REVIEW

Globozoospermia syndrome: An update

Farzaneh Fesahat1 | Ralf Henkel2,3 | Ashok Agarwal3

1Reproductive Immunology Research Center, Shahid Sadoughi University of Abstract Medical Sciences, Yazd, Iran Among the factors involved in male infertility, there is a rare morphology disorder 2 Department of Medical called "globozoospermia" that is classified into total globozoospermia and partial Bioscience, University of the Western Cape, Bellville, South Africa globozoospermia (type I and type II, respectively). This syndrome is primarily char‐ 3American Center for Reproductive acterised by the presence of round‐headed spermatozoa with cytoskeleton defects Medicine, Cleveland Clinic, Cleveland, OH, USA around the nucleus and no acrosome. Current data support the negative correlation between globozoospermia and conventional intracytoplasmic sperm injection (ICSI) Correspondence Ashok Agarwal, American Center for outcomes, revealing the need for the management of patients undergoing assisted Reproductive Medicine, Cleveland Clinic, reproduction technology (ART) through more effective treatment techniques. This Cleveland, OH, USA. Email: [email protected] review highlights the most important characteristics of globozoospermia such as sperm parameters, DNA/chromatin integrity and sperm DNA fragmentation (SDF), as well as genetic features based on the latest knowledge. Additionally, we looked into current progress on fertilisation potential and possible treatment strategies for patients presenting with globozoospermia.

KEYWORDS DNA fragmentation, globozoospermia, human, intracytoplasmic sperm injection, morphology, spermatozoa

1 | INTRODUCTION causes primary male infertility (Singh, 1992). Contrary, men with type II globozoospermia have both normal and round‐headed sperm Among the factors involved in male infertility, there is a rare mor‐ cells with large CDs, which impair motility. In this type of globozo‐ phology disorder called "globozoospermia", first described by Wolff, ospermia, 20%–90% of spermatozoa have no acrosome; therefore, Schill, and Moritz (1976). Globozoospermia is primarily characterised it is also known as partial globozoospermia (Dam et al., 2006, 2011). by the presence of round‐headed spermatozoa without acrosome, The findings of one study demonstrated that partial globozoosper‐ cytoskeleton defects around the nucleus, the absence of a post‐ac‐ mia was actually an oligoasthenoteratozoospermia (OAT) with an rosomal sheath and the separation of nuclear membranes (Figure 1) increased percentage of acrosome‐less spermatozoa (>25%), thus (Han et al., 2017). Another important characteristic of globozoosper‐ showing no uniform pathology (Vicari et al., 2002). mic spermatozoa is their coiled tails (Dam et al., 2006). Furthermore, maturation defects, such as the presence of cytoplasmic droplets (CDs) surrounding the nucleus or intermediate piece, have been fre‐ 2 | PATHOGENESIS AND EPIDEMIOLOGY quently reported (Dam et al., 2006). Singh (1992) classified globozoospermia into type I and type II So far, no clear explanation has been provided for the pathogenesis (Singh, 1992). Accordingly, in type I globozoospermia, also known as of globozoospermia. The morphological characteristics of globozoo‐ total or classic globozoospermia, or true round‐head only syndrome, spermic spermatozoa are formed during spermiogenesis and due to a 100% of the spermatozoa have a small, round and acrosome‐free defect in one or more of the sperm nuclear remodelling mechanisms, head. Since round‐headed spermatozoa are unable to penetrate the especially during the formation of the acrosome and the integra‐ zona pellucida because of having no acrosome, this kind of disorder tion of its vesicles, and sperm elongation (Dam et al., 2006, 2011;

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(a) (b) (c)

(d) (e) (f)

FIGURE 1 Human sperm morphology. Schematic view of normal spermatozoa (a) and sperm cell with globozoospermia (b) primarily characterised by the presence of round‐headed spermatozoa without acrosome. Diff‐Quik (c) and Papanicolaou (d–f) staining were applied to evaluate morphological features for spermatozoa. Stained spermatozoon with normal as well as abnormal round‐headed globozoospermia was seen as dark and colour arrows respectively

Wolff et al., 1976). Other morphological features of globozoosper‐ needed to clarify the epidemiology of both total and partial globozo‐ mic samples include cytoskeleton defects such as the round nucleus, ospermia in different populations. the absence of a posterior acrosomal sheath, separation of nuclear membranes and coiled tails, and the presence of a cluster of differ‐ entiation surrounding the middle piece (Courtot, 1991). The separate 3 | SPERM PARAMETERS development of the acrosome from the Golgi apparatus, the lack of acrosome vesicle fusion and the poor performance of the Golgi ap‐ Some of the human studies indicated that the quality of vari‐ paratus can be mentioned as possible mechanisms for the absence ous semen parameters in globozoospermic men was differentially of acrosomes in globozoospermic spermatozoa (Courtot, 1991). The changed (Table 1). A retrospective study on semen parameters of prevalence of this disorder among infertile men is estimated to be 72 globozoospermic patients collected from all papers published on <0.05%–0.1% in different studies (Kalahanis et al., 2002; Karaca, globozoospermia from 1965 to January 2006 concluded that the Akpak, Oral, Durmus, & Yilmaz, 2015; Kim, Cha, Park, & Gye, 2001). average ejaculatory volume and sperm concentration were normal Round‐headed spermatozoa are also found in normal ejaculates, despite 100% morphological abnormalities in these sperm cells ac‐ but there is no definite threshold for the natural population of these cording to WHO standards (≤1999). However, the average sperm sperm cells in an individual normal ejaculate. In a study conducted motility was decreased by 39% (Dam et al., 2006). In fact, the major‐ on 233 semen samples from infertile men, the percentage of round‐ ity of each included case report in the aforementioned review (Dam headed spermatozoa was estimated to be 0.1%–0.8% (Andrade‐ et al., 2006) showed that the patients with globozoospermia rep‐ Rocha, 2001). Kalahanis et al. reported that globozoospermic sperm resented normal sperm concentrations and semen volume, but the rates were significantly higher in infertile men compared with fertile total sperm motility was in abnormal but admissible range (Larson subjects (2.3 ± 0.5% vs. 0.5% ± 0.1% respectively) (Kalahanis et al., et al., 2001; Vicari et al., 2002). 2002). In contrast, Dam et al. (2011) reported that sperm concentration, Despite little knowledge about the effect of environmental fac‐ progressive motility and normal morphology in globozoospermic tors such as smoking on increasing the number of round‐headed samples were significantly decreased compared with normozoosper‐ sperm cells in individual ejaculates, it is believed that environmental mic controls. In a case–controled study, Eskandiari and colleagues factors might be effective indicators for globozoospermia, especially reported significantly reduced sperm concentration and motility in for the partial type (Rubes et al., 1998). More detailed research is globozoospermic men compared with controls (68.60 and 53.20 vs. FESAHAT et al. | 3 of 13

TABLE 1 List of key findings on semen parameters of globozoospermic men in recent studies

Total motility Semen parameters Volume (ml) Count (106/ml) (%) Normal morphology (%)

Dam et al. (2006) 3.7 (N = 43) 67 (N = 67) 39 (N = 64) 0I Systematic review (WHO ≤1999) Varied based on morphological (Results collected from all papers published on assessmentsII globozoospermia from 1965 to January 2006) Different assessment was used in (N = 75) each study Cases: types I and II Egashira et al. (2009) 2.6 43 32.6 0 (Case report) (reference criteria was not mentioned) Simple light microscopic observa‐ (N = 1) tion was used Case: type I Sermondade et al. (2011) NA 35 30 0 (Case report) (WHO, 2010) Both Harris–Shorr coloration and (N = 1) motile sperm organelle morphol‐ Case: type I ogy examination (MSOME) were used Brahem et al. (2011) 2.1 115 53 0 (Case–control) (WHO, 1992) Methodology was not indicated (N = 2 vs., 20 respectively) Cases: types I and II Dam et al. (2011) 2 (N = 3)I 70I 38I 0A/E & D‐Q, I (Case–control) (WHO, 1999) 2.9 (N = 10)II 21II 22II 28A/E,II, 1D‐Q, II (N = 13 vs., 9 respectively) A/E and D‐Q were used Cases: types I and II Vozdova et al. (2014) 6.2 243 48 0 (Case report) (WHO, 2010) D‐Q and Spermac were used (N = 1) Case: type I Hosseinifar et al. (2015) NA 37.82 18.08 0 (Case–control) ¥ Papanicolaou was used (N = 20 vs., 40 respectively) Case: infertile men with more than 90% globozoospermia Ghasemzadeh et al. (2015) 3.24I 78.07I 33.93I 0I (Case–control) (WHO, 2010) Papanicolaou was used (N = 15 vs., 21 respectively) Case: type I Karaca et al. (2015) 3.2 14 57.33 0 (Case report) (WHO, 2010) D‐Q and TEM were used (N = 1) Case: type I Talebi et al. (2018) 3.3I 86.9I 38.96I 0I (Case–control) (WHO, 2010) 2.6II 38.8II 24.01II 0.19 (min = 0‐max = 3)II (N = 27 vs., 30 respectively) Papanicolaou was used Cases: types I and II Ghazavi et al. (2018) NA 58.5 34.5 0 (Case–control) (WHO, 2010)¥ Methodology was not indicated (N = 6 vs., 1 respectively) Case: type I Shang et al. (2018) 2 37 31 0 (Case series) (WHO, 2010) Acrosomes staining by fluorescein (N = 9) isothiocyanate (FITC)‐labelled Case: type I peanut agglutinin was used

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TABLE 1 (Continued)

Total motility Semen parameters Volume (ml) Count (106/ml) (%) Normal morphology (%)

Eskandari et al. (2018) 3.97 68.60 53.20 0 (Case–control) (WHO, 2010) Papanicolaou was used (N = 30 vs., 22 respectively) Case: type I Tavalaee et al. (2018) 3.73 50.85 37.47 0 (Case–control) (WHO, 2010) Papanicolaou was used (N = 44 vs., 32 respectively) Case: type I Moretti el al. (2019) 4.8 112.66 22.33PM 0 (Case–control) (WHO, 2010) Methodology was not indicated (N = 1 vs., 3 respectively) Case: type I

Note: Spermac: a differential acrosome staining technique to distinguish spermatozoa with acrosomes (Stain Enterprises, Wellington, South Africa); ¥: sperm morphology was evaluated using according to Menkveld, Stander, Kotze, Kruger, & Zyl (1987), & Menkveld, Stander, Kotze, Kruger, & Zyl (1990), and CASA system was used for semen analysis. The mean value is presented for all variables. Abbreviations: I, total globozoospermia (type I); II, partial globozoospermia (type II); N, number of patients; D‐Q, Diff‐Quik staining (Medion Diagnostics AG, Düdingen, Switzerland); PM, progressive motility; A/E, aniline blue and eosin staining; NA, no information.

42.51 and 32.94, respectively), while the mean of semen volume Since spermatozoa require progressive motility to reach the fer‐ was not significantly changed in cases compared with the controls tilisation site in the ampulla of the fallopian tube and penetrate the (p = .97) (Table 1) (Eskandari, Tavalaee, Zohrabi, & Nasr‐Esfahani, cumulus and zona pellucida, sperm motility can be considered as one 2018). A recent study (Tavalaee, Nomikos, Lai, & Nasr‐Esfahani, of the important factors predicting sperm fertilizing potential (Larsen 2018) reported similar findings as explored by Eskandari et al. (2018) et al., 2000). When it comes to globozoospermia, sperm morphology (Table 1). In agreement, our previous studies showed no significant has been found to be the most important parameter as it is indica‐ change in semen volume either in the combined globozoospermia tive of the poor sperm quality and their defects (Sutovsky, Terada, population or in each patient group compared with the controls & Schatten, 2001). Larson et al. (2001) used transmission electron (Ghasemzadeh et al., 2015; Talebi, Ghasemzadeh, Khalili, Halvaei, & microscopy (TEM) and showed that globozoospermic spermatozoa Fesahat, 2018). However, we observed remarkably lower total mo‐ had more vacuoles in their head compared with normal spermatozoa. tility, progressive motility, sperm count and normal morphology in Nardo et al. applied TEM and observed a lack of acrosomal vesicles both types of globozoospermic samples compared with normozo‐ and axonemal structural organisation in round‐headed spermatozoa ospermic controls (p < .001) (Ghasemzadeh et al., 2015; Talebi et (Nardo, Sinatra, Bartoloni, Zafarana, & Nardo, 2002). Further, by al., 2018) (Table 1). In agreement with our results, Hosseinifar et al. using scanning electron microscopy (SEM), they showed that globozo‐ demonstrated significant decreases in sperm concentration, total ospermic spermatozoa had a larger neck, and the head is surrounded motility and normal morphology in patients with globozoospermia by thin cytoplasm. Recently, a case study conducting electron mi‐ compared with the controls (p < .001) (Hosseinifar et al., 2015). croscopy analysis also confirmed the morphological features of glo‐ This finding could be due to two reasons. Firstly, all three stud‐ bozoospermic cells including a round head without acrosome and ies (Ghasemzadeh et al., 2015; Hosseinifar et al., 2015; Talebi et al., with the tail coiled around the nucleus as well as immature chromatin 2018) were performed according to WHO (2010) standards, which (Moretti, Collodel, Salvatici, Belmonte, & Signorini, 2019). Moreover, presented lower cut‐off criteria compared with previously intro‐ some changes in the protein expression and the localisation of pro‐ duced WHO standards (1992 and 1999) (Table 2). Secondly, these teins such as centrin1 in the sperm centriole were detected around researches included 47 oligozoospermic patients, whereas most of the head of globozoospermic spermatozoa in comparison with the the previous studies in this regard were case reports. Accordingly, it controls (Moretti et al., 2019). In addition, in some spermatozoa, twin seems that the findings of the aforementioned studies are more re‐ tails were wrapped around the sperm head (Nardo et al., 2002). liable. However, as shown in Table 1, there are still some recent case According to the literature, it seems that the presence of sperma‐ reports with the normal ranges of average ejaculatory volume sperm tozoa with abnormal morphology (teratozoospermia) and decreased concentration, and total motility according to WHO (2010) standards sperm motility (asthenozoospermia) are two important charac‐ with no control that made it difficult reaching the final conclusion teristics causing remarkable changes in globozoospermic patients (Sermondade et al., 2011; Shang et al., 2018; Vozdova et al., 2014). In (Figure 1). Many clinical reports suggested that sperm morpholog‐ other words, the variety of studies in number and type of samples, ical abnormalities were associated with male infertility (Garrett, Liu, study type and the selective laboratory methods for investigating Clarke, Rushford, & Baker, 2003; Rothmann & Reese, 2007). Besides the sperm parameter may interfere with a comprehensive outcome. the morphological defect of globozoospermic spermatozoa, which FESAHAT et al. | 5 of 13

TABLE 2 Comparison of lower reference limits for the World Health Organization (WHO) in semen parameters

Total motility Normal Total sperm number Semen parameters Volume (ml) Count (106/ml) (%) ­morphology (%) Viability (%) (106/ejaculate)

WHO (1992) 2.0 20 50 30 75 40 WHO (1999) 2 20 50 14 75 40 WHO (2010) 1.5 15 40 4 58 39

Abbreviation: WHO, World Health Organization. is one of the main causes of natural fertility failure, other infertility percentage of chromomycin A3 (CMA3)‐positive spermatozoa and causes in these patients can also lead to poor sperm motility. their correlation in 20 men with total globozoospermia using SCSA, CMA3 staining and fluorescent microscopy. They observed signifi‐ cantly higher levels of DFI and CMA3‐positive spermatozoa in globo‐ 4 | DNA/CHROMATIN INTEGRITY AND zoospermic men compared with controls (p < .001). Hosseinifar and SPERM DNA FRAGMENTATION co‐workers also found a positive correlation between DFI and the percentage of CMA3‐positive spermatozoa in these patients (r = .45, It is known that conventional semen analysis does not reflect the p = .046) (Hosseinifar et al., 2015). These findings suggest that glo‐ fertility status of a man (Cho & Agarwal, 2018; Guzick et al., 2001), bozoospermic men carry abnormal chromatin and sperm DNA com‐ and spermatozoa with normal morphology and motility can also have paction, which is a cause of increased DNA damage in these patients abnormal chromatin/DNA as approximately 8% of infertile men with (Hosseinifar et al., 2015; Talebi et al., 2018) (Table 3). normal sperm parameters have high levels of DNA damage (Agarwal, SDF in mature spermatozoa demonstrated an incomplete nu‐ Bragais, & Sabanegh, 2008). Evidently, sperm DNA integrity is an clear maturation during spermiogenesis (Oosterhuis et al., 2000). To important marker for normal sperm function and can be a better pre‐ clarify the source of SDF, two important hypotheses can be noted: dictor for fertilisation and pregnancy than the common semen pa‐ (a) the abortive apoptosis mechanism and (b) the higher sensitivity rameters (Schulte, Ohl, Sigman, & Smith, 2010). One of the important of deficient chromatin with abnormal condensation rates to oxida‐ applications of DNA integrity assessment is the appropriate sperm tive assaults by reactive oxygen species (ROS) (Perrin et al., 2011). selection in assisted reproduction technology (ART) since the use Apoptosis, or programmed cell death, naturally eliminates abnormal of spermatozoa with abnormal DNA during in vitro fertilisation (IVF) spermatozoa during spermatogenesis (Agarwal & Said, 2003). As a programme leads to a significant reduction in the success rate (Cho result, about 25% of the spermatozoa are removed during this mech‐ & Agarwal, 2018; Zhao, Zhang, Wang, & Li, 2014). Like other sperm anism. The presence of apoptosis markers in semen indicates the morphological abnormalities, globozoospermia is likely to be related presence of abnormal spermatozoa with DNA damage subjected to to DNA damage and an abnormal chromatin structure. Disruption apoptosis (Agarwal & Said, 2003; Agarwal, Virk, Ong, & du Plessis, of the histone replacement by protamines during spermiogenesis, 2014). However, if the elimination process of these sperm cells is higher frequency of sperm DNA fragmentation (SDF), sperm chro‐ not completed due to defective spermatogenesis, apoptotic sperma‐ matin abnormalities and chromatin instability was reported in globo‐ tozoa can escape from the apoptotic elimination and appear in the zoospermic patients (Blanchard, Lescoat, & Le Lannou, 1990; Carrell, ejaculate (Aitken & Koppers, 2011; Muratori et al., 2008). Emery, & Liu, 1999; Vozdova et al., 2014). The second hypothesis for the origin of SDF is the higher sensi‐ Many studies found increased levels of SDF among men with tivity of uncondensed chromatin compared with oxidative assaults globozoospermia versus fertile men using the terminal deoxynucle‐ through reactive oxygen species (ROS) (Agarwal et al., 2014; Aitken otidyl transferase dUTP nick‐end labelling (TUNEL) assay (Baccetti, & Koppers, 2011). Oxidative stress is a condition resulting from an Collodel, & Piomboni, 1996; Eskandari et al., 2018; Ghasemzadeh imbalance between ROS (oxidants) and antioxidant agents in favour et al., 2015; Perrin et al., 2013; Talebi et al., 2018; Vicari et al., of the oxidants. On the other hand, low levels of ROS are essen‐ 2002) (Figure 2). Using the SCD test, Deemeh, Tavalee, Razavi, and tial for normal sperm physiology, hence playing an important role Esfahani (2007) demonstrated increased SDF levels only in one of in cell proliferation, differentiation, gene regulation, protein expres‐ two patients with total globozoospermia. Despite a few contradic‐ sion and thus sperm functions (Agarwal et al., 2014). Moreover, ROS tory reports regarding SDF in globozoospermic men (Larson et al., are essential key players in signalling events in sperm capacitation 2001; Sutovsky et al., 2001), most studies showed a significant in‐ and sperm–oocyte interactions (Makker, Agarwal, & Sharma, 2009). crease in the SDF index of globozoospermic samples compared with However, unphysiologically high levels of ROS directly affect sperm normozoospermic (Brahem, Mehdi, Elghezal, & Saad, 2011; Egashira, DNA (Agarwal et al., 2014). Murakami, Haigo, Horiuchi, & Kuramoto, 2009; Perrin et al., 2011; High levels of ROS can disturb topoisomerase II (TOPO II), an Vicari et al., 2002) (See more references in Table 3). enzyme that is involved in the nuclear remodelling by breaking and Hosseinifar et al. (2015) investigated the DNA fragmentation reconnecting DNA fragments during spermiogenesis, leading to the index (DFI) as well as chromatin condensation by means of the presence of large amounts of DNA nicks in elongating spermatids, and 6 of 13 | FESAHAT et al.

FIGURE 2 Sperm DNA fragmentation (SDF) testing methods. Acridine orange (AO) dye (a) has a metachromatic shift between orange‐ red and green when bound to DNA breaks and normal DNA respectively. Chromomycin A3 (CMA3) staining (b) was used for assessing DNA protamination state related to sperm chromatin condensation. Positive CMA3 (CMA3+) represents abnormal DNA as bright yellow sperm heads and yellowish‐green sperm heads were considered for DNA damage. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL) (c) assay reflects late‐stage sperm apoptotic rate. Spermatozoa with fragmented DNA showed bright green fluorescence (TUNEL+); however, normal DNA was detected in light green (TUNEL−). Toluidine blue (TB) (d) and aniline blue (AB) (e) staining show chromatin condensation with light/unstained mature spermatozoa and blue nuclei of immature spermatozoa with poor chromatin condensation. Figure (f) shows sperm chromatin dispersion (SCD/Halo test) with intact sperm DNA (SCD++), moderate DNA fragmentation (SCD+) and highest DNA fragmentation (SCD−). The partial globozoospermic cells were seen in panels (a), (b), (d) and (e), and total globozoospermic cells were detected in panels (c) and (f) in the comparison view finally SDF (Oosterhuis et al., 2000). Excess ROS can also cause sperm (n = 27), were similar to those reported in a case study by Vozdova mitochondrial DNA damage as well as cytochrome c depletion from et al. (2014), who indicated an increase in the percentage of sper‐ the internal membrane, resulting in sperm cell apoptosis and DNA matozoa with excess histones and in the sperm numbers with lower breakage (Gharagozloo & Aitken, 2011). In general, two factors can chromatin condensation and protamine deficiency (Ghasemzadeh et protect spermatozoa from these destructive effects of ROS: (a) chro‐ al., 2015; Talebi et al., 2018; Vozdova et al., 2014) (Table 3). However, matin condensation with the resulting inaccessibility and protection of there was no remarkable change in sperm numbers with the chro‐ DNA, and (b) the presence of antioxidants in the semen (Greco et al., matin instability in globozoospermic men compared with controls 2005). Therefore, spermatozoa with poor protamination and chroma‐ (Ghasemzadeh et al., 2015; Talebi et al., 2018; Vozdova et al., 2014). tin condensation resulted from defective spermatogenesis are more Since we applied the aniline blue (AB), toluidine blue (TB) and CMA3 susceptible to oxidative stress (Duru, Morshedi, & Oehninger, 2000). stains to detect excessive amounts of histones, low chromatin con‐ Electron microscopy studies, as well as cytochemical assess‐ densation and protamine deficiencies in the testicular phase, it can ments, reported chromatin abnormalities such as protamine defi‐ be concluded that globozoospermia impacts the chromatin conden‐ ciency in the nucleus structure of globozoospermic spermatozoa sation during the testicular phase rather than the epididymal phase (Blanchard et al., 1990; Deemeh et al., 2007; Escalier, 2002; Nardo (Ghasemzadeh et al., 2015; Talebi et al., 2018) (Figure 2). et al., 2002; Taylor et al., 2010; Vicari et al., 2002). Contrary, few Taken together, globozoospermic spermatozoa carry an abnormal studies showed that globozoospermia was not associated with chromatin structure, which negatively impacts on the fertilisation po‐ chromatin structure abnormalities using the SCSA, a quantitative tential of globozoospermic men, causing fertilisation failure in an ART method for determining abnormal sperm chromatin structure, or the programme (Collins, Barnhart, & Schlegel, 2008; Sakkas et al., 1998). sperm chromatin dispersion test (SCD) (Lalonde et al., 1988; Larson It is believed that success in ART cycles depends more on DNA/chro‐ et al., 2001) (Figure 2). Despite different sample sizes, the findings matin integrity than on the sperm morphology, which should be taken of our studies for both, total and partial globozoospermic samples into account when these patients enter the ART programme. FESAHAT et al. | 7 of 13

TABLE 3 List of key findings on sperm DNA fragmentation, chromatin integrity and chromosome aneuploidy of globozoospermic men in recent studies

Variables Results Testing methods References

Sperm DNA fragmentation and Within normal TUNEL Sermondade et al. (2011) chromatin integrity values (6%) (Case report) (N = 1) Case: type I ▲▲I,II TUNEL Brahem et al. (2011) (Case–control) (N = 2 vs., 20 respectively) Cases: types I and II ▲▲I,II TUNEL Perrin et al. (2011) (Case–control) (N = 5 vs., NI respectively) Cases: types I and II

Δ TEM Dam et al. (2011) Poorly condensed (Case–control) chromatin (N = 13 vs., 9 respectively) Cases: types I and II 56.3% AB+ AB Vozdova et al. (2014) spermatozoa TB (Case report) (N = 1) 36.6% TB+ CMA3 Case: type I spermatozoa SCSA 47.7% CMA3+ spermatozoa DFI = 35.3%* HDS = 21.7%* ▲▲I TUNEL Ghasemzadeh et al. (2015) ▲▲I CMA3 (Case–control) (N = 15 vs., 21 respectively) Case: type I ▲▲ CMA3 Hosseinifar et al. (2015) ▲▲ SCSA (Case–control) (N = 20 vs., 40 respectively) Case: infertile men with more than 90% globozoospermia ▲▲I,II TUNEL Talebi et al. (2018) ▲▲I,II SCD (Case–control) ▲▲I,II AO (N = 27 vs., 30 respectively) ▲▲I,II CMA3 Cases: types I and II ▲▲I,II AB ▲▲I,II TB ▲▲I TUNEL Eskandari et al. (2018) ▲▲I CMA3 (Case–control) (N = 30 vs., 22 respectively) Case: type I ▲▲I TUNEL Haghighat et al. (2019) (Case–control) (N = 10 vs., 30 respectively) Case: type I 22.3% AO+ AO Moretti et al. (2019) spermatozoa AB (Case–control) 78% abnormal AB+ TEM (N = 1 vs., 3 respectively) spermatozoa Case: type I Abnormal sperm chromatin conden‐ sation and maturity

(Continues) 8 of 13 | FESAHAT et al.

TABLE 3 (Continued)

Variables Results Testing methods References

Chromosome aneuploidy ▲▲Modest increase FISH Brahem et al. (2011) in sex chromosome For X, Y, 18, 8, 12 (Case–control) aneuploidy and (N = 2 vs., 20 respectively) disomy chr.8 Cases: types I and II Normal karyotype Karyotyping and FISH Sermondade et al. (2011) (46XY) For 13, 18, 21, X, Y (Case report) (N = 1) No aneuploidy Case: type I No increased FISH Perrin et al. (2011) aneuploidy For 13, 18, 21, X, Y (Case–control) Slight increase in (N = 5 vs., NA respectively) disomy for chro‐ Cases: types I and II mosomes X, Y, XY and 21 ▲Sperm ane‐ FISH for X, Y, 8, 18, 21 Vozdova et al. (2014) uploidy for the sex (Case report) (N = 1) chromosomes Case: type I Normal karyotype NA Karaca et al. (2015) (46XY) (Case report) (N = 1) Case: type I

Note: ▲: Increased levels in a case without control. ▲▲: Significant increase compared with controls. Δ Increased levels in case compared with controls with no significant value. Abbreviations: AB, aniline blue to detect excessive amounts of histones; AO, acridine orange; CMA3, protamine deficiencies; DFI & HDS, DNA Fragmentation Index & High DNA stainability with the threshold values of 30% for DFI and 15% for HDS respectively; FISH, fluorescence in situ hybridisation; I, total globozoospermia (type I); II, partial globozoospermia (type II); N, number of patients; NA, no information; SCD, sperm chromatin dispersion test; SCSA, sperm chromatin structure assay for assessing integrity and condensation of sperm chromatin; TB, toluidine blue for evaluat‐ ing low chromatin condensation; TEM, transmission electron microscopy; TUNEL, terminal uridine nick‐end labelling.

5 | GENETIC FEATURES Proteomic analysis of globozoospermic men showed that the SPACA1 protein content was significantly lower in these patients than in normal Some evidence indicated an accumulation of globozoospermia in human spermatozoa (Liao, Xiang, Zhu, & Fan, 2009). Although SPACA1 certain families, suggesting a genetic origin for this condition (Bailey, does not appear to be a major cause of the round‐headed spermato‐ 2002; Carrell, Wilcox, Udoff, Thorp, & Campbell, 2001; Kilani et al., zoa phenotype in humans (Chianese et al., 2015), studies with a larger 2004; Koscinski et al., 2011). However, the pattern of inheritance study population should be done to verify this issue. and the specific genetic defects involved are not well known (Stone, Mutations in three genes, namely spermatogenesis‐associated O'Mahony, Khalaf, Taylor, & Braude, 2000). Thus far, several genes 16 (SPATA16), a protein interacting with C kinase 1 (PICK1) and have been identified in mice, in which the disorder shows a pheno‐ DPY‐19‐like 2 (DPY19L2), were identified as the main genetic cause type similar to that of globozoospermia in humans (Truong, Moses, of globozoospermia (Fujihara, Oji, Larasati, Kojima‐Kita, & Ikawa, Armes, Venter, & Baker, 2003; Xu, Toselli, Russell, & Seldin, 1999). A 2017; Koscinski et al., 2011). Both SPATA16 and PICK1 proteins are protein kinase known as casein kinase II (CK2) seems to be involved in localised in the Golgi apparatus and play a role in the transfer of ves‐ this pathology as mice with any defect in the casein kinase 2 α prime icles from the Golgi apparatus to the acrosome along with acrosome polypeptide gene (Csnk2a2) are infertile (Truong et al., 2003; Xu et biogenesis (Liu, Shi, & Lu, 2010). Furthermore, this study revealed al., 1999). Sperm morphology is similar to that in globozoospermic that Spata16 was required for human sperm formation and its ab‐ humans (Xu et al., 1999). However, in a human study, no mutation sence may lead to male fertility (Fujihara et al., 2017). Moreover, it was detected in Csnk2a2 and Csnk2b genes in patients with globo‐ is now believed that the most common genetic defect observed in zoospermia (Pirrello et al., 2005). human globozoospermia is a homologous deletion in the DPY19L2 Fujihara et al. (2012) reported that disruption of the sperm acro‐ gene (Coutton et al., 2012; ElInati et al., 2012; Tavalaee et al., 2018). some‐associated 1 gene (Spaca1) in mice can lead to abnormal sperm DPY19L2 is located on chromosome 12 with 109.66 kb in length. Its morphology similar to human globozoospermia, resulting in infertility. product contributes to the stabilisation of the acrosome, promotion Spaca1 is a membrane protein that is presented in mammalian sperma‐ of the acroplaxome anchorite to the nuclear membrane, mediation tozoa including the human and plays an important role in sperm–oocyte of normal sperm morphology formation and the elongation of the interaction and fusion in a way that is disorder causes the disappear‐ sperm head and acrosome formation (ElInati et al., 2012; Harbuz ance of acrosomes in mature spermatozoa (Fujihara et al., 2012). et al., 2011; Pierre et al., 2012; Sha et al., 2018). FESAHAT et al. | 9 of 13

By screening the DPY19L2 variants during a cohort study, all nine development in mammals after fertilisation, triggering the calcium Chinese globozoospermic patients showed genetic deficiencies such oscillations causes resumption of meiosis (Nasr‐Esfahani, Deemeh, as deletion or point mutations in the DPY19L2 gene (Shang et al., & Tavalaee, 2010), sperm nuclear decondensation, the use of ma‐ 2018). Ghazavi, Peymani, Hashemi, Ghaedi, and Nasr‐Esfahani (2018) ternal RNA, the formation of the male and female pronucleus, the found DPY19L2 deletion in 75% of men with globozoospermia who onset of DNA synthesis and eventually embryonic cleavage (Nasr‐ attended Isfahan Fertility and Infertility Center as a referral centre Esfahani et al., 2010). Normal spermatozoa trigger these events by for globozoospermia in Iran. As sufferers are routinely screened for oocyte activation via the release of a cytosolic protein factor called DPY19L2 deletion in this centre and according to a recent report by oscillin. This protein was proposed as a hamster homologue to the Ghazavi et al. (2018), it has been suggested that embryos derived human SAOAF causes the calcium influx required for oocyte activa‐ from couples in consanguineous marriages with globozoospermia tion during fertilisation (Montag, Parrington, Swann, Lai, & Van der should be screened for gender or DPY19L2 deletion in order to de‐ Ven, 1998). Rybouchkin, Dozortsev, Pelinck, De Sutter, and Dhont crease the occurrence of globozoospermia in future progeny. Recent (1996) analysed the oocyte activation capacity of round‐headed findings have clarified that DPY19L2 might have a key role in nuclear human spermatozoa by injection into mouse oocytes and showed chromatin organisation in the sperm nucleus via its interaction with that oscillin was not expressed in round‐headed spermatozoa. These the nuclear lamina (NL) (Abdelhedi et al., 2019). The impact of the NL authors discovered that fertilization, and cleavage rate increased and its associated proteins during human spermiogenesis has been following ICSI in the presence of oocyte activating factors, such as emphasised (Paci et al., 2017). Hence, a lack of NL maturation and calcium chloride or calcium ionophore A23187 (Rybouchkin et al., differences in expression and localisation of lamin chromatin part‐ 1996). Furthermore, Khakpour, Sadeghi, Tavalaee, Bahadorani, & ners such as lamin b1, b3 emerin, LAP2α, BAF and BAF‐L in globo‐ Nasr‐Esfahani (2019) obtained similar results and concluded that zoospermia could be a good justification for chromatin defects and ICSI using artificial oocyte activation (AOA) led to a high fertilisation lower fertility potential and embryo developments after ICSI than rate in globozoospermic patients with low expression of phospholi‐ controls (Paci et al., 2017). pase C zeta (PLC ζ) as one of the important sperm factors involved in oocyte activation (Tavalaee et al., 2018). Accordingly, several preg‐ nancies (Banker, Patel, Joshi, Shah, & Goyal, 2009; Ghazavi et al., 6 | FERTILISATION POTENTIAL AND 2018; Kim et al., 2001; Taylor et al., 2010) and even live birth (Banker TREATMENTS et al., 2009; Ghazavi et al., 2018) were reported in such patients. Therefore, if oocyte activation is performed correctly, globozoosper‐ 6.1 | Conventional ICSI mic sperm cells may support fertilisation and embryo development. Some evidence showed that in total globozoospermia, normal fertil‐ Secondly, although oocyte activation in globozoospermic pa‐ ity was not occurred in IVF after insemination of human oocytes due tients following ICSI may lead to fertilisation and cleavage, it might to the inability of the round‐headed spermatozoa to bind or pen‐ not necessarily lead to pregnancy because of the poor sperm chro‐ etrate the zona pellucida (Liu et al., 1995). Before the introduction matin condensation and high levels of SDF. Poor chromatin packag‐ of intracytoplasmic sperm injection (ICSI), there was no therapeu‐ ing or DNA damage can lead to failure of nuclear decondensation, tic treatment for globozoospermic men (Palermo, Joris, Devroey, & inability to support the early stages of embryo development, which Van Steirteghem, 1992). Therefore, ICSI was revolutionary in the will consequently lead to fertilisation failure after ICSI (Sakkas et al., treatment of these patients. The first successful ICSI using globo‐ 1998). Therefore, it is believed that ART success depends more on zoospermic spermatozoa was reported in a patient with total glo‐ DNA and chromatin integrity than on sperm morphology (Avendaño bozoospermia (Liu et al., 1995). Subsequently, many other reports & Oehninger, 2011; Simon, Zini, Dyachenko, Ciampi, & Carrell, 2017). indicated successful achievement of pregnancy with globozoosper‐ Thirdly, sperm aneuploidies can also affect ICSI outcomes. mic spermatozoa after using ICSI (Battaglia, Koehler, Klein, & Tucker, Increased aneuploidy rates often occur in acrocentric (13, 14, 15, 1997; Heindryckx, Van der Elst, De Sutter, & Dhont, 2005; Stone 18 and 21) and sex chromosomes in globozoospermic rather than in et al., 2000). However, due to the reduced ability of globozoosper‐ fertile men (Carrell et al., 1999, 2001; Dirican, Isik, Vicdan, Sozen, & mic spermatozoa to activate oocytes as well as their poor chromatin Suludere, 2008; Kochhar & Ghosh, 2018). However, these findings condensation and high SDF, the overall fertilisation rate still remains have not yet been confirmed by other studies (Rybouchkin et al., lower than 50% (Dam et al., 2011; Larson et al., 2001; Stone et al., 1996; Vicari et al., 2002). Molecular cytogenetic and genetic aspects 2000). Current data support the negative correlation between glo‐ of globozoospermia were reviewed by Perrin until 2013. Here, we bozoospermia and conventional ICSI outcomes, suggesting the need attempted to show the key findings on chromosome aneuploidy for advanced management of globozoospermic patients undergo‐ of globozoospermic men in recent studies (Table 3). Altogether, it ing ART using more effective treatment techniques (Chianese et al., seems that globozoospermia is responsible for the presence of 2015). higher aneuploidies in spermatozoa, and globozoospermic patients In general, there are three reasons for the failure of ICSI in these have a normal 46 XY karyotype, and thus, their spermatozoa can be patients: Firstly, the oocyte‐activating factor (SAOAF) is inhibited used in ART (Karaca et al., 2015). or missing. Since oocyte activation is the first stage of embryo 10 of 13 | FESAHAT et al.

Apart from the three above‐mentioned reasons, new study has AOA may help overcome infertility in patients with total globozoo‐ introduced an altered profile of lipid and fatty acid oxidation as a pos‐ spermia. Considering the strong relationship between the DPY19L2 sible reason responsible for fertilisation failure in ICSI (Moretti et al., gene and globozoospermia, it can be concluded that the most rel‐ 2019). Moretti et al. investigated the sperm centriole status as one evant genetic factor in patients with either total or partial globo‐ of a required structure for human fertilisation as well as the impact zoospermia is this gene. Therefore, screening of DPY19L2 gene can of fatty acid oxidation in globozoospermia. They found an increased be considered as a helpful strategy for a pre‐implantation genetic nonenzymatic fatty acid oxidation and centrin1 alteration in a case diagnosis of men with total or partial globozoospermia in order to with total globozoospermia compared with the three fertile controls improve the ICSI results (clinical pregnancy and live birth) and fam‐ (Moretti et al., 2019). However, other researches need to provide a ily counselling, especially for populations with high rates of consan‐ further understanding of the relationship not only between lipid per‐ guineous marriages. oxidation and male infertility but also in regard to globozoospermia.

ACKNOWLEDGEMENTS

6.2 | New treatment techniques along with ICSI The author (Farzaneh Fesahat) wishes to thank Late Mr.

Successful ART outcomes by resorting to some new treatment pro‐ Ghasemzadeh, who has motivated her to write this research tocols/techniques along with ICSI, such as ICSI with AOA using cal‐ manuscript. cium ionophore, may help overcome male infertility in patients with total globozoospermia (Abadi, Tavalaee, Shahverdi, & Nasr‐Esfahani, CONFLICT OF INTEREST 2016; Karaca et al., 2015; Kochhar & Ghosh, 2018; Shang et al., 2018; Tavalaee et al., 2018). Some candidate genes such as truncated The authors declare that they have no financial conflict of interest. Kit, PLC ζ and post‐acrosomal sheath WW domain‐binding protein (PAWP) are found to be responsible for oocyte activation during ORCID fertilisation (Abadi et al., 2016; Haghighat et al., 2019; Tavalaee & Nasr‐Esfahani, 2016). Significantly lower expression profiles of Farzaneh Fesahat https://orcid.org/0000-0002-3743-4449 these genes and their corresponding proteins in globozoospermic Ralf Henkel https://orcid.org/0000-0003-1128-2982 men have been reported, suggesting a beneficial effect of AOA re‐ Ashok Agarwal https://orcid.org/0000-0003-0585-1026 sulting in higher fertilisation rates after conventional ICSI (Abadi et al., 2016; Haghighat et al., 2019; Tavalaee & Nasr‐Esfahani, 2016; Taylor et al., 2010). In this regard, a review by Chansel‐Debordeaux, REFERENCES Dandieu, Bechoua, and Jimenez (2015) suggests recombinant PLC Abadi, M.‐K.‐D., Tavalaee, M., Shahverdi, A., & Nasr‐Esfahani, M. H. , as a promising biological treatment to improve the reproductive ζ (2016). Evaluation of PLCζ and PAWP expression in globozoospermic outcome in ART. Furthermore, the motile sperm organelle morphol‐ individuals. Cell Journal (Yakhteh), 18(3), 438. ogy examination (MSOME) technique can be another useful tool for Abdelhedi, F., Chalas, C., Petit, J. M., Abid, N., Mokadem, E., Hizem, S., evaluating sperm morphology at high magnification (greater than … Dupont, J. M. (2019). Altered three‐dimensional organization of sperm genome in DPY19L2‐deficient globozoospermic patients. 6000x) and more detail (Bartoov et al., 2002; Larsen et al., 2000). To Journal of Assisted Reproduction and Genetics, 36(1), 69–77. https​:// the best of our knowledge, MSOME can better increase the success doi.org/10.1007/s10815-018-1342-y of ART outcome in globozoospermic patients than routine sperm Agarwal, A., Bragais, F. M., & Sabanegh, E. (2008). Assessing sperm func‐ morphological analysis for ICSI using standard microscopic tech‐ tion. Urologic Clinics of North America, 35(2), 157–171. https​://doi. org/10.1016/j.ucl.2008.01.012 niques (Talebi et al., 2018). Agarwal, A., & Said, T. M. (2003). Role of sperm chromatin abnormali‐ ties and DNA damage in male infertility. Human Reproduction Update, 9(4), 331–345. https://doi.org/10.1093/humup​ d/dmg027​ Agarwal, A., Virk, G., Ong, C., & du Plessis, S. S. (2014). Effect of oxidative 7 | CONCLUSION stress on male reproduction. The World Journal of Men's Health, 32(1), 1–17. https​://doi.org/10.5534/wjmh.2014.32.1.1 In general, it can be stated that globozoospermia decreases sperm Aitken, R. J., & Koppers, A. J. (2011). Apoptosis and DNA damage in quality, specifically motility, and normal morphology. Secondly, human spermatozoa. Asian Journal of Andrology, 13(1), 36. https​:// doi.org/10.1038/aja.2010.68 there is a strong positive correlation between globozoospermia and Andrade‐Rocha, F. (2001). Sperm parameters in men with suspected defective sperm chromatin condensation and DNA damage. Poor infertility. Sperm characteristics, strict criteria sperm morphology chromatin packaging and/or damaged DNA can be the main reasons analysis and hypoosmotic swelling test. The Journal of Reproductive for the inability of round‐headed spermatozoa for normal nuclear Medicine, 46(6), 577–582. Avendaño, C., & Oehninger, S. (2011). DNA fragmentation in morpho‐ decondensation and embryo development. This may explain the low logically normal spermatozoa: How much should we be concerned fertilisation rates after ICSI in globozoospermic patients. Applying in the ICSI era? Journal of Andrology, 32(4), 356–363. https​://doi. new treatment techniques along with conventional ICSI such as org/10.2164/jandrol.110.012005​ FESAHAT et al. | 11 of 13

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