Genetic basis of acephalic spermatozoa syndrome, and intracytoplasmic sperm injection outcomes in infertile men: a systematic scoping review

Abstract

Purpose

Acephalic spermatozoa syndrome (ASS) is known as a severe type of teratozoospermia, defined as semen composed of mostly headless spermatozoa that affect male fertility. In this regard, this systematic review aimed to discuss gene variants associated with acephalic spermatozoa phenotype as well as the clinical outcomes of intracytoplasmic sperm injection (ICSI) treatment for the acephalic spermatozoa-associated male infertility.

Methods

A systematic search was performed on PubMed, Embase, Scopus, and Ovid databases until May 17, 2020. This systematic scoping review was reported in terms of the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) statement.

Results

Twenty articles were included in this systematic review. Whole-exome and Sanger sequencing have helped in the identification of variants in SUN5, PMFBP1, BRDT, TSGA10, DNAH6, HOOK1, and CEP112 genes as possible causes of this phenotype in humans. The results of the ICSI are conflicting due to both positive and negative reports of ICSI outcomes.

Conclusion

ASS has a genetic origin, and several genetic alterations related to the pathogenesis of this anomaly have been recently identified. Notably, only SUN5 and PMFBP1 mutations are well-known to be implicated in ASS. Accordingly, more functional studies are needed to confirm the pathogenicity of other variants. ICSI could provide a promising treatment for acephalic spermatozoa-associated male infertility. Besides the importance of sperm head-tail junction integrity, some other factors, whether within the sperm cell or female factors, may be involved in the ICSI outcome.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10815-020-02008-w.

Introduction

Human infertility is described as the couple’s inability to conceive a child following a year of having regular unprotected intercourse [1]. Approximately 15% of couples are affected by infertility worldwide, and about 50% of them are due to male factor infertility [2]. Male factor infertility is mainly caused by the deterioration of qualitative and quantitative sperm parameters [3]. The typical spermatozoon is composed of a head and flagellum or a tail. The tail of the sperm can be divided into three parts as follows: the midpiece, principal piece, and end piece (Fig. 1). Teratozoospermia can be defined as the presence of spermatozoa with normal morphology lower than the cut-off values determined in the ejaculate by the World Health Organization (WHO) [6].

Fig. 1.

A Schematic diagram of normal human spermatozoa. A normal spermatozoon is composed of the head and flagellum or tail. The neck of the sperm connects the head and tail. The head comprises of the nucleus and acrosome. The tail of the sperm is divided into the midpiece, principal piece, and end piece. Both of the proximal and distal centrioles are enclosed by the segmented columns. Also, the capitulum, a curved plate-like structure, encloses the proximal centriole cranially. The capitulum associates with the basal plate at the implantation fossa to link connecting pieces to sperm heads. Distal ends of the segmented columns attach to the outer dense fibers (ODFs). ODFs associate with outer microtubule doublets of the axoneme. The mitochondrial sheath is formed by the accumulation of mitochondria around the axoneme in the midpiece [4, 5]. Autodesk 3ds Max was used to create this artwork

Acephalic spermatozoa syndrome (ASS) is a rare severe type of teratozoospermia. Correspondingly, ASS is defined as semen composed of mostly headless spermatozoa affecting male fertility [7, 8]. Due to the presence of both detached heads and tails in the semen, this syndrome is also termed as the decapitated and decaudated sperm defect [9]. Accurate information on the morphological features of the decaudated and decapitated sperm cells was obtained by the ultrastructural examinations [9–13]. Notably, head and tail separation occurs between the proximal and distal centriole [11] or along with the midpiece [9] or most frequently occurs between the nucleus and the centriolar region [12–15]. Some loose heads, lacking the implantation fossa and the basal plate, have been reported to be present in the ejaculate [14–16]. Some earlier ultrastructural studies have suggested various mechanisms for the formation of acephalic spermatozoa [12]. It was shown that the overproduction of a membranous vesicle system by the Golgi complex during the spermatid stage between the centrioles and acrosome may affect the sperm head-tail attachment [10]. Defects in the mechanisms of migration and positioning of the tail on the caudal pole of the nucleus or some nuclear defects interfering with the formation of implantation fossa and basal plate were also proposed to be a probable cause of the decapitated cells [16]. The reduction or the absence of pericentriolar granular material leads to non-developing segmented columns in the connecting piece. Therefore, this can probably cause dissociation of the distal centriole from the proximal centriole in the first steps of spermatid differentiation. Subsequently, the sperm head and tail separately develop [11].

Head-tail separation can take place either in the testis or in the epididymis. In the testicular separation, the heads remain attached to the residual bodies at spermiation, which consequently go through phagocytosis by the Sertoli cells. Therefore, the ejaculate contains many tails and few loose heads [12, 16]. In the epididymal separation, although the defect originates in the testicular region, detached heads and tails are equally present in the ejaculation [9]. Some of the detached tails have forward motility, and the rest of them are immotile or have non-progressive motility [10, 11, 14, 16]. The hypoosmotic swelling and zona-free Hamster egg tests have established that most of the headless tails are functionally normal as tails [15]. In initial reports, familial incidences of acephalic spermatozoa were observed in men and bulls. Accordingly, this supports the possibility of a genetic origin for ASS [8, 9, 12, 14, 17].

Although several microscopic examinations provided a detailed morphological description of the decapitated and decaudated spermatozoa, little was known about the pathogenesis of this anomaly. Genetic alterations associated with acephalic spermatozoa have been recently identified (Fig. 2). Thereby, this systematic review was performed with the purpose of discussing both gene variants associated with acephalic spermatozoa phenotype as well as the clinical outcomes of intracytoplasmic sperm injection (ICSI) treatment for acephalic spermatozoa-associated male infertility.

Fig. 2.

Timeline of discoveries in human acephalic spermatozoa syndrome. Accurate information on the morphological features of the decaudated and decapitated sperm cells was obtained by the ultrastructural examinations in earlier studies. Recent descriptive data obtained from whole-exome sequencing (WES) and Sanger sequencing in humans has revealed acephalic spermatozoa syndrome-associated gene mutations. SUN5, Sad1 and UNC84 domain containing 5; BRDT, bromodomain testis associated; PMFBP1, polyamine modulated factor 1 binding protein 1; TSGA10, testis-specific gene antigen 10; DNAH6, dynein axonemal heavy chain 6; HOOK1, hook microtubule tethering protein 1; CEP112, centrosomal protein 112. Autodesk 3ds Max was used to create this artwork

Methods

This systematic scoping review was reported in terms of the PRISMA statement [18].

Search strategy

A literature search was made by the use of PubMed, Embase, Scopus, Ovid, and ProQuest (thesis) databases with no restrictions up to May 17, 2020. Moreover, Google scholar was another source of search in order to find unpublished literatures. The search strategy was based on the following keywords: “Acephalic spermatozoa” or “Acephalic Spermatozoa Syndrome” or “Headless Spermatozoa” or “Headless tail” or “decapitated spermatozoa” AND “Whole exome sequencing” or “WES” or “Next generation sequencing” or “NGS” or “Sanger sequencing” AND “Intracytoplasmic sperm injection” or “ICSI” AND “gene” or “Mutation.” The search strategy for the above-mentioned databases is shown in Supplementary Table S1.

Study selection

Articles were exported into EndNote X8 software, and duplicates were then removed. The title, abstract, and full text of the final papers were reviewed, and the relevant articles were enrolled. Non-English studies were excluded from the study. In addition, in the present systematic scoping review, research articles discussing gene variants associated with acephalic sperm phenotype and studies in which ICSI treatment was performed for infertile men with ASS were included as eligible articles. Searching and screening the articles were separately performed by two researchers.

Data extraction

The details of the enrolled articles including author, publication year, identified genes, the identified cDNA and amino acid alterations, and ICSI outcomes are shown in Table 1.

Table 1.

Details of included studies

Author, year	Studied patients	Origin	Percentage of acephalic spermatozoa (%)	Identified genes	Identified cDNA alteration (amino acid alteration)	Mutations in men treated with ICSI	ICSI treatment outcome	Reference
Zhu et al. 2016	ASS = 17 Control DNA sample = 100	China	94.9–99.7	SUN5	c.824C>T (p.Thr275Met), c.1066C>T (p.Arg356Cys), c.485T>A (p.Met162Lys), c.381delA (p.Val128Serfs*7), c.781G>A (p.Val261Met), c.216G>A (p.Trp72*), c.1043A>T (p.Asn348Ile), c.425+1G>A, c.851C>G (p.Ser284*), c.340G>A (p.Gly114Arg)	_	_	[19]
Sha et al. 2018	ASS = 15	Fujian, China	50–80	SUN5	c.381delA (p.Val128Serfs*7)	_	_	[20]
Elkhatib et al. 2017	ASS = 3 Infertile men without the acephalic sperm phenotype = 150	North Africa (Algeria)	~ 80–99	SUN5	GRCh38 - chr20:32995761_32990672delinsTGGT p.Leu143Serfs*30	_	_	[21]
Shang et al. 2018	ASS = 3	China	78.95–95.45	SUN5	c.475C>T (p.Arg159*)	_	_	[22]
Fang et al. 2018	ASS = 3 Couples underwent ICSI = 8	China	95.2–98.8	SUN5	c.829C>T (p.Gln277*), c.1067G>A (p.Arg356His), c.211+1 insGT (p.Ser71Cysfs11*)	P1: c.824C>T/c.381delA P2: c.216G>A/c. 1043A>T P3: c.851C>G P4: c.425+1G>A/c.1043A>T P5: c.829C>T/c.1066C>T P6: without SUN5 mutations P7: without SUN5 mutations P8: without SUN5 mutations	Couple 1: one twin Couple 2: one baby Couples 3, 4, and 5: ongoing pregnancies Couples 6, 7, and 8: became pregnant	[23]
Liu et al. 2020	ASS = 3	China	~ 70	SUN5	c.772C>T (p.Arg258Cys)	P: c.772C>T	Transplantable embryos or blastocysts were not achieved	[24]
			~ 100	PMFBP1	c.361C>T (p.Gln121Ter), c.2089-1G>T (p.Ile697Leufs*257)	_	_
			~ 99	TSGA10	c.1739A>C (p.Gln580Pro)	P: c.1739A > C	Became pregnant
Li et al. 2017	ASS = 1	China	99.5	BRDT	c.2783G>A (p.Gly928Asp)	_	_	[25]
Sha et al. 2019	ASS = 2	China	91–92	PMFBP1	c.2561_2562del (p.Lys854Arg fs*5), c.327T>A (p.Tyr109Ter)	_	_	[26]
Zhu et al. 2018	ASS = 23 Control DNA sample = 100 Couples underwent ICSI = 3	China	95.3–98.2	PMFBP1	c.1462C>T (p.Gln488*), c. 2404C>T (p.Gln802*), c. 2725C>T (p.Arg909*), c.2092delG (p.Ala698Profs*7)	P1: c.1462C>T P2: c.1462C>T P3: c.2404C>T	Couple 1: one baby Couple 2: one baby Couple 3: became pregnant	[27]
Sha et al. 2018	ASS = 1 Couples underwent ICSI = 1	China	99	TSGA10	c.211delG (p.Ala71His fs*12)	P: c.211delG	No pregnancy occurred	[28]
Ye et al. 2020	ASS = 1	China	~ 83	TSGA10	c.545dupT (p.Ala183Serfs*10)	_	_	[29]
Li et al. 2018	ASS = 1 Couples underwent ICSI = 1	China	30	DNAH6	c.2454A>T (p.Glu818Asp), c.7706G>A (p.Arg2569His)	P: c.2454A>T/c.7706G>A	No pregnancy occurred	[30]
Chen et al. 2018	ASS = 7 Control samples = 160	China	> 95	HOOK1	c.848T>C (p.Gln286Arg)	P1: without this loci mutation P2: c.848T>C P3: without this loci mutation P4: c.848T>C	Couple 1: failed pregnancy Couple 2: one baby Couple 3: two babies Couple 4: two babies	[31]
Sha et al. 2020	ASS = 2	China	92.4–95.6	CEP112	c.496C>T (p.Arg166Ter), c.2074C > T (p.Arg692Trp), c.2104C>T (p.Arg702Cys)	_	_	[32]
Shang et al. 2017	Couples underwent ICSI = 2	China	P1: 95.7 P2: 96.6	SUN5	_	P1: c.824C>T (p.Thr275Met) P2: c.340G>A (p.Gly114Arg)	Couple 1: one baby Couple 1: one baby	[33]
Sha et al. 2017	Couples underwent ICSI = 3	China	P1: 99 P2: 30 P3: 99	_	_	_	No pregnancy occurred	[34]
Rondanino et al. 2015	Couples underwent ICSI = 3	Egypt, Algeria, Europe	P1: 94.34 P2: 85.32 P3: 63.78 P4: 19.56	_	_	_	No successful pregnancy	[35]
Gambera et al. 2010	Couples underwent ICSI = 2	Italy	P1: ~ 80 P2: ~ 80	_	_	_	Couple 1: one baby Couple 2: no pregnancy occurred	[36]
Porcu et al. 2003	Couples underwent ICSI = 2	North Africa (Algeria)	numerous	_	_	_	Couple 1: one baby Couple 2: three babies	[37]
Saias-Magnan et al. 1999	Couples underwent ICSI = 1	_	Numerous	_	_	_	No pregnancy occurred	[38]

P patient

Results

The articles (a total of 523) were obtained from the above-mentioned electronic databases, and 196 studies have finally remained after removing duplicates. According to the inclusion criteria, of the remaining papers, 20 articles were recognized as eligible for the current systematic scoping review. The essential details of the studies are summarized in Table 1. A flow diagram of the study is shown in Fig. 3.

Fig. 3.

A flow diagram of the studies included in this systematic scoping review

Genetic alterations associated with human acephalic spermatozoa syndrome

Recent descriptive data obtained from WES and Sanger sequencing in humans have revealed ASS-associated gene mutations (Table 2).

Table 2.

Genetic variants discovered in infertile men with acephalic spermatozoa by WES and Sanger sequencing technologies

Identified genes	cDNA alteration	Amino acid alteration	Exon	Mutation	Zygosity in affected individuals	Domain	Reference
SUN5	c.824C>T	p.Thr275Met	11	Missense	Homozygous/compound heterozygous	SUN domain	[19]
	c.1066C>T	p.Arg356Cys	13	Missense	Compound heterozygous	SUN domain	[19]
	c.485T>A	p.Met162Lys	8	Missense	Compound heterozygous	Coiled-coil domain	[19]
	c.381delA	p.Val128Serfs*7	6	Frameshift	Homozygous/compound heterozygous	_	[19, 20]
	c.781G>A	p.Val261Met	11	Missense	Compound heterozygous	SUN domain	[19]
	c.216G>A	p.Trp72*	4	Nonsense	Compound heterozygous	N-terminus	[19]
	c.1043A>T	p.Asn348Ile	13	Missense	Compound heterozygous	SUN domain	[19]
	c.425+1G>A	_	7	Splice site	Compound heterozygous	_	[19]
	c.851C>G	p.Ser284*	11	Nonsense	Homozygous	SUN domain	[19]
	c.340G>A	p.Gly114Arg	5	Splice site	Homozygous	Transmembrane domain	[19]
	GRCh38 - chr20:32995761_32990672delinsTGGT	p.Leu143Serfs*30	8	Frameshift	Homozygous	_	[21]
	c.475C>T	p.Arg159*	8	Nonsense	Compound heterozygous	coiled-coil domain	[22]
	c.829C>T	p.Gln277*	11	Nonsense	Compound heterozygous	SUN domain	[23]
	c.1067G>A	p.Arg356His	13	Missense	Homozygous	SUN domain	[23]
	c.211+1 insGT	p.Ser71Cysfs11*	3	Frameshift	Compound heterozygous	N-terminus	[23]
	c.772C>T	p.Arg258Cys	NM	Missense	Homozygous	SUN domain	[24]
BRDT	c.2783G>A	p.Gly928Asp	19	Missense	Homozygous	P-TEFb binding	[25]
PMFBP1	c.2561_2562del	p.Lys854Arg fs*5	17	Frameshift	Homozygous/compound heterozygous	Smc domain	[26]
	c.327T>A	p.Tyr109*	4	Nonsense	Compound heterozygous	N-terminus	[26]
	c.1462C>T	p.Gln488*	11	Nonsense	Homozygous	Smc domain	[27]
	c. 2404C>T	p.Gln802*	16	Nonsense	Homozygous	Smc domain	[27]
	c. 2725C>T	p.Arg909*	19	Nonsense	Homozygous/compound heterozygous	C-terminus	[27]
	c.2092delG	p.Ala698Profs*7	15	Frameshift	Compound heterozygous	Smc domain	[27]
	c.361C>T	p.Gln121*	NM	Nonsense	Compound heterozygous	N-terminus	[24]
	c.2089-1G>T	p.Ile697Leufs*257	NM	Frameshift	Compound Heterozygous	Smc domain	[24]
TSGA10	c.211delG	p.Ala71Hisfs*12	7	Frameshift	Homozygous	Phosphodiesterase	[28]
	c.1739A>C	p.Gln580Pro	18	Missense	Homozygous	COG4372 domain	[24]
	c.545dupT	p.Ala183Serfs*10	8	Frameshift	Homozygous	Phosphodiesterase	[29]
DNAH6	c.2454A>T	p.Glu818Asp	15	Missense	Compound heterozygous	_	[30]
	c.7706G>A	p.Arg2569His	48	Missense	Compound heterozygous	_	[30]
HOOK1	c.848T>C	p.Gln286Arg	10	Missense	Heterozygous	Central coiled-coil domain	[31]
CEP112	c.496C>T	p.Arg166*	5	Nonsense	Homozygous	_	[32]
	c.2074C>T	p.Arg692Trp	20	Missense	Compound heterozygous	Coiled-coil domain	[32]
	c.2104C>T	p.Arg702Cys	20	Missense	Compound heterozygous	Coiled-coil domain	[32]

NM not mentioned, Smc structural maintenance of chromosomes

SUN5

SUN5 (Sad1 and UNC84 domain containing 5) [MIM: 613942], also called SPAG4L, is a well-known gene whose variants have been demonstrated to cause ASS [19–22]. The SUN domain proteins contain a transmembrane domain in the N-terminus as well as a conserved C-terminal domain. Furthermore, they are known as a part of the LINC complexes (linkers of the nucleoskeleton to the cytoskeleton). Accordingly, this complex links the nucleoskeleton to the cytoskeleton [39, 40]. SUN5 encodes a testis-specific protein, which is a transmembrane protein in the inner nuclear membrane. SUN5 contains an N-terminus in the nucleoplasmic region, a transmembrane domain, a coiled-coil domain, and a SUN domain. The SUN domain is within the C-terminus locating in the perinuclear space. SUN5 is localized in the spermatozoon head-tail junction, which may take part in the attachment of the sperm tail to the head [19–21, 33].

Since 2016, 16 SUN5 variants associated with ASS have been identified using WES and Sanger sequencing in infertile men with more than 50% acephalic sperms in their semen. Segregation analysis and family pedigrees have suggested an autosomal recessive mode of inheritance. It was indicated that these 16 reported mutations in the SUN5 gene include missense, nonsense, intronic mutations, frameshift, and deletions that were localized to the transmembrane helix, and conserved coiled-coil and the SUN domain [19–24]. Experimental studies have been performed on some missense and nonsense reported variants in mice. Considering the evolutionary conservation of SUN5 protein between mouse and human, these findings are hypothesized to be similar to those of humans. Accordingly, the variant within the splice site led to a premature stop codon in the SUN5 mRNA, which consequently caused a truncated SUN5 protein leading to the loss of the SUN domain. The mutation within the transmembrane domain caused the aggregation of SUN5 proteins mostly in the cytoplasm, rather than in the nuclear envelope and thus SUN5 dysfunction. Additionally, the variants located in the coiled-coil domain caused a truncated protein with a disrupted coiled-coil domain as well as the alteration of the SUN5 secondary structure. The variants in the SUN domain affected the normal distribution of the SUN5 and the secondary structure, which led to misfolded proteins [22].

Consequently, these identified mutations might decrease SUN5 expression levels or might affect the localization or secondary structure of SUN5 protein. Moreover, they might also disturb the interaction of SUN5 with its partners like some KASH (Klarsicht, ANC-1, and Syne homology) or LINC proteins in the perinuclear space. In conclusion, these observations provided some evidence indicating that the loss of SUN5 function could lead to the failure of head-tail connection formation [19–24].

The histological examination performed in Sun5 knockout mice revealed that testis weight was not different from wild-type mice. The testicular component was similar in these two groups, and also sperm concentration was not significantly different in the caudal epididymis. It is noteworthy that the basal plate-capitulum-segmented column complex was partially connected to the nuclear envelope. The nuclear envelope and the coupling apparatus connection were then separated during the spermatid elongation with no functional SUN5. Afterward, although headless tails and a few normal heads entered into the epididymis, most heads remained in the seminiferous epithelium. In other words, the sperm head and flagellum separation occurred in the spermiation phase before releasing into the epididymis [33]. In conclusion, all these findings indicated that SUN5 is required for sperm head and tail integrity.

BRDT

BRDT (bromodomain testis associated) [MIM: 602144] encodes a testis-specific protein. Accordingly, it contains two canonical bromodomains involved in chromatin remodeling [41, 42]. Bromodomains interact with the modified histones and recognize acetylated lysines in histones as well as nonhistone proteins. In this regard, numerous chromatin and transcription-related proteins, such as histone acetyltransferases and chromatin remodeling factors, contain bromodomains. Therefore, due to these bromodomains, BRDT is able to interact with acetylated histone H4 tails. BRDT also plays a structural role in the condensation of acetylated chromatin [42], and the BRDT protein is a transcriptional regulator [43, 44]. Li et al. in their study in 2017 using WES reported a missense mutation in a highly conserved Gly928 in the BRDT gene in an infertile man who had 99.5% acephalic sperm cells. Based on the segregation analysis and family pedigrees, an autosomal recessive mode of inheritance was suggested [25]. The missense variant is localized within the P-TEFb binding domain in the C-terminal of the BRDT protein. In addition, the P-TEFb binding domain mediates the interaction with the transcription elongation factor [25, 43, 45]. Therefore, it was found that this variant may alter the transcriptional activities of the BRDT protein. Although this variant cannot affect the expression level of the BRDT protein, it can alter the expression of 899 genes involving in intracellular transport, DNA metabolic processes, RNA transport, cell cycle, and RNA splicing processes. Notably, some of the up-regulated genes are involved in the endosome to the trans-Golgi network process. The overproduction of a membranous vesicle system by the Golgi complex between the centrioles and acrosome might affect the attachment of the sperm proximal centrioles and the nucleus [10, 25]. Hence, the up-regulation of these genes might be associated with the head and tail detachment [25]. The identified missense variant was characterized as “probably damaging” and “disease-causing” based on the in silico computations. Thereafter, we used InterVar to interpret the variant in terms of the American College of Medical Genetics (ACMG) guidelines. Correspondingly, this variant was classified as a variant with unknown significance (VUS) [46].

PMFBP1

PMFBP1 (polyamine modulated factor 1 binding protein 1) [MIM: 618085], which encodes a sperm tail-associated protein (STAP), is highly expressed in adult testis [26, 47]. PMFBP1 is located in the head-tail junction of normal spermatozoa, which joins the coupling apparatus to the sperm nuclear envelope. PMFBP1 is positioned in the medium region between SUN5 and SPATA6 in mouse and human spermatozoa. Altogether, these three proteins are not known to have any interaction with each other. PMFBP1 cooperates with SUN5 in joining the sperm head to the tail. Three research groups have identified eight variants in the PMFBP1 gene in ASS patients with more than 91% headless spermatozoa using WES and Sanger sequencing. The identified mutations were nonsense and frameshift. The nonsense mutations, located in the N-terminus of the PMFBP1, resulted in a premature termination codon leading to a short N-terminus lacking the C-terminus. The nonsense and frameshift mutations located in the C-terminus of the PMFBP1 indicated the importance of the C-terminal region in the connection of the sperm head to the coupling apparatus. Notably, this protein was lost in acephalic spermatozoa, and the break site may be located in the neck region [24, 26, 27].

The same phenotype was observed in Pmfbp1-knockout mice that was generated using the CRISPR/Cas9 technology. The histological examination revealed that testis size and weight were not different between wild-type and Pmfbp1-knockout mice. In addition, seminiferous tubules morphology was normal in Pmfbp1-deficient mice, which contained all the seminiferous epithelium components. Although total sperm count in the caudal epididymis of Pmfbp1-knockout mice was not different from the wild type, the sperm heads were absent. It was observed that the coupling apparatus and sperm nucleus connection were destroyed during the spermiogenesis process in the Pmfbp1-deficient mice [26, 27]. Another important finding of this study was the misregulation of 159 proteins in sperm from Pmfbp1 KO mice. The biological process of most of the up-regulated proteins was related to Golgi vesicle transport. In consistent with this finding, Li et al. have proposed that the overproduction of the Golgi complex might be involved in the head-tail detachment [25, 26]. Briefly, PMFBP1 is required for joining the coupling apparatus to the sperm head. Also, the truncating mutations in this gene are associated with the ASS [24, 26, 27].

TSGA10

The protein encoded by the TSGA10 (testis-specific gene antigen 10) [MIM: 607166] gene seems to play a role in spermatogenesis [48, 49]. The N-terminus of the TSGA10 protein is located in the principal piece. Also, the C-terminus is located in the midpiece to the centrosome [28]. WES and Sanger sequencing validation have revealed two frameshift and one missense mutations within TSGA10 in three infertile men who presented more than 83% of headless spermatozoa in the ejaculate. Autosomal recessive inheritance was suggested based on family pedigree [24, 28, 29]. The homozygous missense variant was identified in a highly conserved region within the C-terminal COG4372 domain [24]. The frameshift variants are located in the phosphodiesterase domain within the N-terminus, resulting in a stop codon gain. Therefore, the entire C-terminus of TSGA10 protein is missing, and the transcript might be degraded due to the nonsense-mediated decay. Loss of the C-terminus might affect the TSGA10 protein function, consequently impair the midpiece structure, and lead to acephalic sperm cells [28, 29]. Although TSGA10 was localized in the centriole and midpiece of normal spermatozoa, the full-length TSGA10 protein was not found in the acephalic spermatozoa. The mitochondrial sheath was also impaired, and a partial mitochondrial sheath was present in both head and tail of the detached sperm, which indicated the breakage in the midpiece. Besides, central pairs and the peripheral doublet microtubules of the axoneme were completely absent in the acephalic sperm cells. In summary, the absence of TSGA10 led to head-tail detachment in the midpiece, which might cause axoneme impairment [28, 29].

Further investigations revealed that the loss of the TSGA10 did not alter the SUN5 and PMFBP1 localizations. In patients with PMFBP1 mutation, normal positioning of the SUN5 was observed; however, the localization of TSGA10 was altered. Furthermore, the localization of PMFBP1 in the Sun5-null spermatozoa was impaired in the sperm head due to the disruption of Sun5. As a result, SUN5 and TSGA10 were located in the upstream and downstream of PMFBP1, respectively [26, 27, 29]. According to the in silico prediction tools, variants identified in TSGA10 are predicted to be disease-causing. We have also used VarSome to interpret the variants in terms of the ACMG guidelines. Although the missense variant was classified as VUS, the two frameshift variants were classified as pathogenic [50].

DNAH6

DNAH6 (dynein axonemal heavy chain 6) [MIM: 603336], which encodes the axonemal dynein heavy chain, belongs to the family of dynein proteins. Dyneins are microtubule-associated motor protein complexes that convert the chemical energy into mechanical energy through a mechanochemical cycle to generate force and movement on microtubules in some biological processes such as ciliary movement and intracellular transport. Moreover, DNAH6 expression is high in tissues containing cilia like testis, ovary, brain, and lung [51, 52]. Mutations in DNAH6 was shown to be associated with multiple morphological abnormalities of the sperm flagella (MMAF) [51] and azoospermia [53]. Compound heterozygous variants of the DNAH6 gene were also identified using WES in an infertile man with 69% globozoospermia and 30% acephalic spermatozoa in the semen. In this regard, a recessive mode of inheritance was suggested based on pedigree analysis. The DNAH6 protein was located in the neck region of the normal spermatozoa. However, both of the DNAH6 protein and mRNA were lost in the studied patient. Probably, these variants finally resulted in the DNAH6 mRNA decay. According to the in silico computational algorithms, the p.Glu818Asp, and p.Arg2569His variants were predicted to be benign and probably damaging, respectively [30]. The variants were then classified as VUS in terms of the ACMG guidelines [46]. Correspondingly, further studies are still required to reveal DNAH6 function in spermatogenesis as well as its disruption effect on spermatozoa head-tail detachment [30, 53, 54].

HOOK1

HOOK1 (hook microtubule tethering protein 1) [MIM: 607820] encodes a member of the hook family proteins. HOOK1 is a coiled-coil protein taking part in the manchette-nucleus connection as well as in the correct arrangement of microtubular structures in the haploid germ cell [31, 55, 56]. A novel missense mutation was found in the central coiled-coil domain of the HOOK1 gene in an infertile man with more than 95% acephalic spermatozoa in his semen, which can probably be associated with severe decapitated and decaudated spermatozoa syndrome. Also, the HOOK1 protein structure was predicted to be probably damaged. This mutation is suggested to have a dominant heredity, based on the performed family pedigree analysis. In headless tails, the structure of axoneme, proximal centrioles, and mitochondria ridge were normal; however, the implantation fossa and the basal plate in the tailless heads were incomplete and disorganized [31]. Deletion of exons 10 and 11 of the Hook1 gene was identified in the abnormal spermatozoon head shape (azh) mutant mouse models that presented decapitated and decaudated spermatozoa, which affected the sperm head and flagellum attachment [56].

CEP112

CEP112 (centrosomal protein 112) is highly expressed in testes. The centrosomal protein encoded by CEP112 belongs to the CEP family proteins and contains a coiled-coil domain. Centrosomal proteins are known as the components of the centrosome involved in centriole biogenesis, cell cycle progression, and spindle-kinetochore assembly control. The centriole is important for the sperm head-tail attachment [32, 57]. Two missense and one nonsense mutations were identified using WES and Sanger sequencing in the CEP112 gene in two infertile men with more than 90% headless sperms in their semen. These variants were predicted to be deleterious by in silico analysis. The nonsense mutation causes a premature stop codon, which consequently leads to a truncated protein lacking the entire coiled-coil domain as well as the absence of CEP112 in the patient. The missense mutations are located within the coiled-coil domain and reduce CEP112 expression. It is notable that the altered amino acid properties in Arg692 and Arg702 may affect both CEP112 folding and function. CEP112 was found in the centrioles of the normal spermatozoon. However, CEP112 was hardly detectable in the head and tail of sperm from the patient carrying the nonsense variant. Moreover, its expression was also very weak in the spermatozoa of the patient carrying the missense mutations. These loss-of-function mutations in CEP112 might affect the coiled-coil domain, function, and stability of the protein. According to these findings, CEP112 was reported to be essential for the sperm head-tail attachment [32]. Also, all these variants were classified as VUS in terms of the ACMG guidelines [46].

Intracytoplasmic sperm injection as a promising treatment for acephalic spermatozoa-associated male infertility

Up to now, many studies have focused on the impact of sperm morphology on the clinical outcomes of natural conception and assisted reproductive technology (ART). Although earlier findings were conflicting, most of the recent studies revealed a minor clinical effect of sperm morphology on ART and natural pregnancy outcomes [58–63]. It was also indicated that the isolated teratozoospermia is not associated with a decreased likelihood of pregnancy after IVF with or without ICSI [62]. Also, normal sperm morphology is not a predictor of either fertilization rates or pregnancy rates of ICSI cycles [64–67].

Four infertile men with mutations in SUN5 who presented numerous acephalic spermatozoa in the semen could become fathers by recruiting sperm with the attached head and tail via ICSI [21, 33, 37]. Fang et al. in their study in 2018 compared the ICSI results of five infertile ASS men harboring SUN5 mutations, three ASS men with no SUN5 mutations, and nine men with MMAF. In this regard, three ongoing pregnancies and three live births of couples with acephalic spermatozoa-associated male infertility with SUN5 mutations were finally achieved. However, one of the children had left ear dysplasia with a mild hearing loss. Partners of all ASS men without SUN5 mutations became pregnant using motile spermatozoa with the head and tail still joined even with an abnormal junction. In addition, ICSI results were not different among the ASS patients; however, fertilization rates were significantly lower in infertile men with MMAF compared to the other two groups [23]. Despite the positive results obtained in previous studies, a recent ICSI attempt on a single egg from a couple that the male carried SUN5 missense mutation could not lead to transplantable embryos or blastocysts [24]. By injecting abnormal head-tail joined spermatozoa into the oocytes in three couples in whom men harbored PMFBP1 nonsense mutations, all the partners became pregnant, and live births were finally reported [27]. Two ICSI attempts in two couples in whom men were harboring TSGA10 mutations consequently resulted in one successful pregnancy and one failure due to the poor embryo quality [24, 28]. In two infertile men with ASS carrying the HOOK1 mutation, live births were achieved using ICSI. However, ICSI attempts in two ASS patients without the HOOK1 mutation resulted in two live births only in one of the couples. These findings indicated that although HOOK1 might involve in the head-tail connection, it may not be critical in sperm fertility [31]. The spouse of a male with ASS harboring DNAH6 variants could not become pregnant [30].

In some other studies, ICSI failed to result in live births in infertile patients presented with the decapitated spermatozoa syndrome with no identified mutations. It can be due to the reason that syngamy and cleavage did not occur, implantation was unsuccessful, or because the embryo quality was poor [12, 34–36, 38]. Also, sperm nuclear alterations such as chromosome abnormalities, DNA fragmentation, and chromatin hypocondensation were proposed to be involved in pregnancy failure after ICSI in four infertile men with decapitated spermatozoa [35]. It is noteworthy that the human sperm centrosome organizes the sperm aster and controls the first mitotic divisions after fertilization. Thus, defective centrosomes may be responsible for fertilization arrest, which leads to ICSI failure [36, 68–71]. The sperm cell in an EDSS (easily decapitated sperm syndrome) patient was decapitated during the immobilization before performing the ICSI process. Intriguingly, by adequate sperm immobilization and appropriate positioning of the sperm midpiece (comprising normal centrosomes), proximal to the sperm head, one live birth was achieved. Interestingly, this finding suggests that donor centrosome therapy may be conceivable in future [72].

The results of the ICSI are conflicting due to both positive and negative reports on the ICSI outcomes. Besides the importance of sperm head-tail junction integrity, some other factors whether within the sperm cell or female factors may be involved in the outcome of ICSI [38]. Successful pregnancies mentioned in couples who underwent ICSI treatment can provide some clues on the subject that ICSI may help overcoming infertility in the couples seeking to parent a child.

Conclusions

Next-generation sequencing has helped discovering some causative gene mutations in infertile men related to quantitative anomalies (azoospermia and oligospermia), motility anomalies (asthenospermia and flagellar abnormalities impairing movement), and morphological anomalies (teratozoospermia, macrozoospermia, and globozoospermia) [73]. ASS, which is a rare type of teratozoospermia, can be defined as semen mostly composed of headless spermatozoa affecting male fertility. This is a recent field, with the first identification of mutations reported in 2016 for SUN5. Earlier findings in this regard have presumed a genetic origin for this phenotype, but the definitive proof was lacking. Recent descriptive data obtained from WES and Sanger sequencing have helped identifying variants in SUN5, PMFBP1, BRDT, TSGA10, DNAH6, HOOK1, and CEP112 genes that can probably be the causes of this phenotype in humans thus far. Only SUN5 and PMFBP1 mutations are well-known to be implicated in ASS. In addition, there is only preliminary evidence that the reported variants in BRDT, DNAH6, HOOK1, and CEP112 are the causes of acephalic spermatozoa in infertile men. In this review, we utilized ACMG guidelines to interpret the pathogenicity of these missense variants, which were classified as VUS. None of these missense variants have been modeled in the mouse by knockout. In this regard, more functional studies are still needed to confirm the pathogenicity of these missense variants. WES findings suggest this technology to help identify further novel ASS associated variants and as an important tool in genetic counseling for infertile men with ASS.

ICSI could be known as a promising treatment for acephalic spermatozoa-associated male infertility. The presence of the nucleus in the head of the sperm cell should be checked in ICSI attempts to avoid selecting the acephalic sperm cell. Once the ASS underlying pathogenic mutations have been identified in a man presented with ASS, the mutations of the same gene should also be screened in his partner to prevent born of children inheriting recessive homozygous or compound heterozygous mutations. Couples seeking a treatment for infertility using the ART should also be cautious regarding the increased risk of congenital malformations as well as imprinting disorders among the offspring born using ART [74–76].

Loss of function of Odf1, Odf2, Spata6, Hook1, and Spatc1l have been found to cause acephalic spermatozoa, which subsequently led to male infertility in mice [56, 77–81]. The reported gene mutations in mice with similar phenotypes may also be potentially associated with human male infertility due to ASS. Additionally, genetically manipulated laboratory animals could also be used to dissect the possible molecular functions of novel human gene mutations associated with acephalic spermatozoa.

Screening these known mutations in infertile men seeking medical advice can provide a diagnosis that clarifies the genetic causes of sperm defects. Moreover, investigating further proteins located in the sperm head-tail coupling apparatus could uncover the molecular mechanisms underlying sperm decapitation that lead to male sterility. Male infertility is an interesting topic in the clinic, so further investigations of the etiology of this phenotype will hopefully help exploring more relative genetic abnormalities and also help developing more potent treatments with fewer side effects.

Data availability

Data sharing not applicable—no new data generated. The data used to support the findings of this study are included in the article.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

This is a systematic scoping review. No ethical approval is required.

Code availability

Not applicable.