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102 Journal of Reproduction & Contraception doi: 10.7669/j.issn.1001-7844.2014.02.0102 2014 Jun.; 25(2):102-118 E-mail: [email protected] Homogeneous Microscopic Abnormalities in Sperm Morphology and Immotility as A Cause of Male Infertility Delia Acevedo León 1* , Josep Ventura Gayete 1 , Carmen Aguado Muñoz 2 , Carmen Carda Batalla 3 , Miguel Armengot Carceller 4 , Jerónimo Forteza Vila 5 1. Department of Biochemistry, Clinical Analysis, Universitary Hospital Dr. Peset, 46017 Valencia, Spain 2. Cytology Unit, Prince Felipe Research Centre (CIPF), 46012 Valencia, Spain 3. Pathology, Medical School, University of Valencia, 46010 Valencia, Spain 4. General and Universitary Hospital, Rhinology Unit, ENT Department, Surgery Department, University of Valencia, 46014, Valencia, Spain 5. Valencian Institute of Pathology (IVP), Catholic University of Valencia San Vicente Mártir, 46003 Valencia, Spain Objective To study the identification of the cause of specific sperm abnormalities. Methods Two adult men with specific alterations in sperm morphology causing 100% immobility were included in this study. The study of sperm used: transmission electron microscopy (both patients); apoptotic markers, DNA fragmentation test and fluorescence in-situ hybridization (patient 1) and immunocytochemistry study of sperm flagellum using anti-β tubulin antibodies and ciliary activity test (patient 2). Results Increased DNA fragmentation (52.6%) and apoptosis biomarkers were detected in patient 1, and loss of the central pair of microtubules in patient 2 (‘9+0’ axoneme); the nasal ciliary activity was normal. Conclusion Results suggest an apoptotic origin of the abnormalities in the sperm from patient 1 and dysplasia of the fibrous sheath in patient 2. Key words: apoptotic changes; dysplasia of fibrous sheath; midpiece thickening; necrospermia; sperm immotility Corresponding author: Delia Acevedo León; Tel: + 34-961622529; Fax: + 34-961622537; E-mail: [email protected] Sperm abnormalities such as morphological and motility disorders are associated with male infertility. The two main causes of sperm immotility are necrozoospermia (the presence of non-viable spermatozoa) and ultrastructural abnormalities of sperm tails. The causes of necrospermia are varied, including testicular disorders, endocrinopathies, defects in androgen

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Page 1: Homogeneous Microscopic Abnormalities in Sperm Morphology ... · Homogeneous Microscopic Abnormalities in Sperm Morphology and Immotility as A Cause of Male Infertility Delia Acevedo

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Journal of Reproduction & Contraception doi: 10.7669/j.issn.1001-7844.2014.02.0102

2014 Jun.; 25(2):102-118 E-mail: [email protected]

Homogeneous Microscopic Abnormalities in Sperm

Morphology and Immotility as A Cause of Male Infertility

Delia Acevedo León1*, Josep Ventura Gayete1, Carmen Aguado Muñoz2, Carmen Carda

Batalla3, Miguel Armengot Carceller4, Jerónimo Forteza Vila5

1. Department of Biochemistry, Clinical Analysis, Universitary Hospital Dr. Peset, 46017 Valencia, Spain

2. Cytology Unit, Prince Felipe Research Centre (CIPF), 46012 Valencia, Spain

3. Pathology, Medical School, University of Valencia, 46010 Valencia, Spain

4. General and Universitary Hospital, Rhinology Unit, ENT Department, Surgery Department, University of

Valencia, 46014, Valencia, Spain

5. Valencian Institute of Pathology (IVP), Catholic University of Valencia San Vicente Mártir, 46003 Valencia,

Spain

Objective To study the identification of the cause of specific sperm abnormalities.

Methods Two adult men with specific alterations in sperm morphology causing 100%

immobility were included in this study. The study of sperm used: transmission electron

microscopy (both patients); apoptotic markers, DNA fragmentation test and fluorescence

in-situ hybridization (patient 1) and immunocytochemistry study of sperm flagellum

using anti-β tubulin antibodies and ciliary activity test (patient 2).

Results Increased DNA fragmentation (52.6%) and apoptosis biomarkers were

detected in patient 1, and loss of the central pair of microtubules in patient 2 (‘9+0’

axoneme); the nasal ciliary activity was normal.

Conclusion Results suggest an apoptotic origin of the abnormalities in the sperm from

patient 1 and dysplasia of the fibrous sheath in patient 2.

Key words: apoptotic changes; dysplasia of fibrous sheath; midpiece thickening; necrospermia;

sperm immotility

Corresponding author: Delia Acevedo León; Tel: + 34-961622529; Fax: + 34-961622537;

E-mail: [email protected]

Sperm abnormalities such as morphological and motility disorders are associated with

male infertility. The two main causes of sperm immotility are necrozoospermia (the presence

of non-viable spermatozoa) and ultrastructural abnormalities of sperm tails. The causes of

necrospermia are varied, including testicular disorders, endocrinopathies, defects in androgen

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biosynthesis, toxins, drugs, chronic systemic diseases, infections and epididymal function or

storage defect[1,2].

With regard to the morphological alterations, there are two main types of

teratozoospermia: heterogeneous non-specific anomalies in different sperm components (the

most frequent type) or a very homogeneous microscopic systematic sperm defect pattern

which is present in most spermatozoa[3]. The latter can affect the sperm head or flagellum,

with abnormalities in the head-neck region including varying degrees of misalignment, up to

the most extreme defect resulting in acephalic spermatozoa[4].

In this paper, we present two cases of sperm immotility, one with thickening of the

midpiece and anomalous head-neck attachment causing some headless sperm with severe

necrospermia features we had not previously seen (patient 1), and another with short and

thick tails , findings compatible with previously reported cases of dysplasia of the fibrous

sheath (DFS) and/or primary ciliary dyskinesia (PCD) (patient 2). To reach the diagnosis of

both cases, basic semen analysis and biochemical study of the seminal plama were insufficient.

For this reason, ultrastructural studies and other techniques as appropriate were added:

In case 1, special stainings, fluorescence in situ hybridisation (FISH) and apoptosis

markers on sperm were performed. FISH was carried out to estimate the proportion of

aneuploidy and structural aberrations present in his ejaculate[5]. DNA fragmentation is the

most characteristic feature of apoptosis, which is caused by the endogenous DNA degrading

endonucleases activated by apoptotic responses, for example, caspase-associated cell

signalling[6]. The DNA damage correlates well with the reproductive potential of sperm[7].

In case 2, to confirm the diagnosis of DFS and/or PCD, immunocytochemistry study of

sperm flagellum and nasal ciliary beat and cellular rotation test were performed. Regarding

flagellar structure, all eukaryotic cilia and flagella possess a central bundle of microtubules

(formed by the polymerization of α- and β-tubulin), called the axoneme, which consists of

nine doublet microtubules surrounding a central pair of singlet microtubules (the distinctive

‘9+2’ microtubule arrangement). The fibrous sheath is a cytoskeletal structure encasing the

axoneme and other dense fibres in the principal piece of sperm tail. It consists of two

longitudinal columns linked to axonemal doublets 3 and 8[8].

PCD is a congenital disease in which the respiratory cilia are immotile, dysmotile, or

both[9]; resulting in a range of chronic clinical manifestations such as bronchitis, rhino-sinusitis

and otitis media, situs inversus[10]. These anomalies are observed in the flagella of spermatozoa

of most patients, causing male infertility. Among the systematic sperm tail defects, DFS has

been particularly well characterised[11,12]. It is a disorder related to PCD in which there is

both widespread disarray of the sperm fibrous sheath and axonemal ciliary defects. In these

cases the majority of spermatozoa are rigid, short, thick, and immotile and the fibrous sheath

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appears hypertrophic, discontinuous, and disorganised. Affected sperm also have perturbed

axonemal structures which might include part or complete absence of dynein arms, lack the

central microtubule pair or radial spokes, or disorganised microtubules[13].

Materials & Methods

Patients

Two adult men with total sperm immotility and characteristic microscopic abnormalities

were included in this study. Both patients were referred to the fertility laboratory after 2 and

3 years of primary infertility history respectively. They had not undergone any medical or

surgical treatments in the three months prior to semen examination. A signed informed-

consent form was obtained from the two patients included in the study, and the project was

approved by the ethics committee at the University Hospital Dr. Peset before starting.

Patient 1

A 37-year-old man who had a previous genitourinary infection caused by Enterococcus

faecalis was treated with Ciprofloxacin, which resolved three months later. Hormonal

studies were normal except for slightly increased follicle-stimulating hormone (FSH) levels.

He underwent scrotal ultrasound and a small cyst in the left testicle was identified. Family

history: the patient has a brother with diagnosed azoospermia who was not included in this

study.

Patient 2

A 39-year-old man with no history of genitourinary infections and negative semen

bacteriological cultures. Hormonal serum screening and his somatic phenotype were normal.

The patient had chronic secretory otitis media in his left middle ear and he underwent myrin-

goplasty in 2010. Given this history, a nasal ciliary activity test was performed in order to

check the PCD diagnosis.

Semen analysis in both patients

Semen samples were collected by masturbation after 4 d of sexual abstinence. For light

microscopy five independent samples from each patient were studied over a 6 12-month

period and analysed within 60 min of collection. Semen parameters were evaluated according

to World Health Organization guidelines[14]. A Makler® (Sefi-Medical Instruments, Haifa,

Israel) chamber was used to determine the sperm concentration and motility by direct

observation with a phase contrast microscope Eclipse E400 Nikon® (Tokyo, Japan). Semen

samples were stained with Sperm VitalStain® (Nidacon International AB, Mölndal, Sweden)

and the percentage of the corresponding dead (stained) and living (unstained) cells was

calculated. The morphological study was performed after methanol fixation and subsequent

staining with modified Giemsa (Quick Panoptic®, QCA, Amposta, Spain), classifying the

sperm as normal or abnormal.

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As part of the biochemical analysis, fructose and citric acid concentrations in the

seminal plasma were determined enzymatically (BioSystems S.A., Barcelona, Spain).

Control reference values were: citrate greater than 24 μmol/ejaculate; fructose greater than

13 μmol/ejaculate. For the ultrastructural studies an aliquot from the same samples used in

the biochemical analysis was studied using transmission electron microscopy (TEM). The

semen samples were fixed in 2.5% glutaraldehyde (buffered in pH 7.4, 0.1 mol/L Sorensen

phosphate solution). The tissue was then post-fixed for 2 h in 1% osmium tetroxide in buffer,

and after dehydration with graded concentrations of acetone, the tissue blocks were embedded

in Epon 812® (TAAB Lab, England). Semi-thin Epon sections (1 μm) were stained with

toluidine blue as a light microscope control and were trimmed for ultrastructural study.

Ultra-thin sections were cut with a diamond knife using a Reichert-Jung ultramicrotome®

(Leica, Illinois, USA), contrasted with uranyl acetate and lead citrate, and examined in a Jeol

Jem 1010® (Tokyo, Japan) electron microscope operated at 60 kV.

Patient 1

Meiotic segregation was explored by FISH using a mix of DNA probes for chromo-

somes 13, 18, 21, X, and Y. The studies were performed using alpha-satellite probes for the

chromosome centromeres and were labelled as follows: 18p11.1 q11 with the aqua

fluorochrome, Xp11.1 Q11.1 with the green fluorochrome, Q11.1 Yp11.1 with the red

fluorochrome, and unique sequence probes for identifying the 13q14 and 21q22.13 q22.2

loci with green and red fluorochromes. Automatic study of 3 808 spermatozoa was performed

with the Metafer MetaCyte system® (MetaSystems, Altlussheim, Germany). Reference

values from the control population: nullisomy less than 3%; disomy and diploidy less than 2%.

For DNA fragmentation analysis, terminal dUTP nick-end labelling (TUNEL), which labels

breaks in ssDNA and dsDNA using an in situ cell death fluorescein detection kit® (Roche

Applied Science, Basel, Switzerland), was performed. The DNA fragmentation index

from 1 266 spermatozoa was obtained. Patient sperm was also used for TUNEL positive

and negative hybridization controls which were achieved following in situ cell death fluorescein

detection kit® instructions (1 000 spermatozoa from each control). Reference value of the

fertile population was < 20% positive.

For analysis of apoptosis markers, cell pellets were lysed on ice in Laemmli’s buffer

(62.5 mmol/L Tris-HCl pH 6.8, 2% sodium dodecyl sulphate, 5% β-mercaptoethanol, 10%

glycerol, and 0.01% bromophenol blue) for 30 min in presence of complete protease inhibitor

cocktail (Roche Applied Science, Basel, Switzerland), they were then boiled for 5 7 min at

100 and subjected to Western blotting analysis. Crude extracts (10 μg) were separated by

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% polyacryla-

mide gel), using human fibroblasts treated for 5 min with ultraviolet light to induce apoptosis

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as positive control. Polyvinylidene difluoride (PVDF) membranes were blocked with 5% dry

milk powder dissolved in tris-buffered saline (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L

NaCl) for 1 h at room temperature and then incubated with different primary antibodies:

Annexin V (Abcam, Cambridge, UK), Poly (ADP-ribose) polymerase (PARP1), and

Caspase-3 (Cell Signalling, Danvers, USA), all at a 1 1 000 dilution, overnight at 4 ,

followed by incubation with the corresponding IgG-horseradish peroxidase (HRP) conjugated

secondary antibody (at a 1 3 000 dilution) for 2 h at room temperature. Blots were visualised

using a Lumi-Light Western blotting detection kit (Roche Applied Science, Basel, Switzerland).

In order to determine the composition of the material deposited on the midpiece, Oil

Red O (an oil-soluble dye for staining lipids) and periodic acid-schiff (to detect polysaccharides

such as glycogen, and mucosubstances such as glycolipids, glycoproteins, and mucins) were

used. The Papanicolaou stain was also used to complement the morphological study. A

karyotype and Y-chromosome study were also performed on whole blood to search for

microdeletions in the azoospermia factor region Yq11 (AZF) and a, b, and c loci were tested.

Patient 2

An immunocytochemistry study of sperm flagellum using mouse monoclonal anti-βtubulin antibodies was performed. Cells on glass slides were fixed for 5 min with methanol at

20 . After this, the cells were washed twice with phosphate buffered saline (PBS) and

blocked with 4% foetal bovine serum/PBS for 30 min. Immunostaining was subsequently

carried out, by incubating the cells overnight at 4 with mouse monoclonal anti-β tubulin

antibodies (at a 1 200 dilution; ab6046, Abcam, Boston, USA). The next day, the cells

were washed twice with PBS and incubated in the dark with goat anti-rabbit secondary

antibody Alexa Fluor 488 (at a 1 500 dilution; A11078, Invitrogen, Carlsbad, USA) for 1 h

at room temperature. The slides were mounted and observed using an Observer Z.1®

fluorescence microscope (Carl Zeiss, Jena, Germany).

To analyse β tubulin, cell pellets were lysed on ice in Laemmli’s buffer for 30 min in the

presence of a mixture of several protease inhibitors with broad inhibitory specificity (complete

protease inhibitor cocktail, Roche Applied Science, Basel, Switzerland), boiled for 5 7 min at

100 and subjected to Western blotting. Mouse monoclonal antibodies which recognise βtubulin (1 1 000 dilution; ab6046, Abcam, Cambridge, UK) and anti-actin (1 10 000

dilution; A-2066, Sigma-Aldrich, St. Louis, USA) were used. Blots using anti-rabbit (1

5 000 dilution) or anti-mouse (1 10 000 dilution) primary antibodies were probed with IgG-

HRP conjugated secondary antibody, and visualised using a Lumi-Light Western blotting

detection kit (Roche Applied Science, Basel, Switzerland).

To check for the presence of impaired respiratory ciliary motility and characteristic of

PCD, nasal ciliary beat frequency and beat pattern were tested, and cellular rotation test

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was performed using high resolution digital high-speed video imaging (DHSV), as previously

described[15]. Samples of ciliary airway epithelial cells were obtained from the middle nasal

concha using curettage without local anaesthesia, in a period during which acute infection

was absent. The tissue sample was immersed in 1 ml of Dulbecco’s modified Eagle’s

medium (DMEM, Cambrex, Walkersville, USA) supplemented with 10% fetal calf serum

(FCS), 2 mmol/L glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml), and turned

over until partial dissolution occurred. Then, 150 μl of the nasal epithelium biopsy cells were

plated onto tissue culture plates (12-well Corning® Costar® 3513, New York, USA), previously

coated with 0.5% gelatin to promote adherence of the ciliated cells. The ciliary beat

frequency was measured at room temperature (23 27 ) within 30 min of biopsy.

Samples were tested using the high resolution digital high-speed video imaging (DHSV)

technique with a Nikon Eclipse TS100® (Tokyo, Japan) microscope equipped with a 40

Nikon phase-contrast objective, providing a final optical gain of 400 . A Multimetrix® XA3051

power source was used with transillumination provided by visible and red-filtered 150 W

halogen lamps. The light path was directed through the multi-image module to a charge-

coupled device (CCD) camera (JAI CV-A33 CL® digital quad high-speed progressive scan

camera) with a resolution of 649 (horizontal) 494 (vertical) pixels and a maximum

resolution velocity of 120 frames per second. Video signals were digitised and processed

with an HP Workstation (xw6200, Xeon 3.4 GHz). The normal range of nasal cilia beat

frequency is between 9 and 13 Hz. Cellular rotation test: the test is positive when an isolated

nasal ciliary cell spontaneously turns on itself quickly and continuously with a regular pattern

and ciliary beat frequency. If the movement is dyskinetic, the ciliary cell does not rotate

(negative test).

Results

Table 1 summarises the main features of the semen samples from both patients

included in this study.

Patient 1

The morphological study revealed thickening of the midpiece (Figure 1A), more

evident in the vitality stain (Figure 1B), indicating that 85% of spermatozoa were affected,

with 12% being decapitated forms (Figure 1B). Most spermatozoa were dead (99%). The

ultrastructural TEM study showed many headless spermatozoa (Figure 2A), high density

deposits (Figure 2B), and disorganisation in the midpiece. Normal mitochondria could not be

distinguished (Figure 2C). The FISH assay on semen samples from patient 1 (Tables 2,3)

showed an abnormally increased percentage of nullisomic spermatozoa (above 3%), affecting

the five chromosomes studied. Diploidy 18/X/Y was identified in more than 2% of spermatozoa.

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Table 1 Semen characteristics and biochemical analysis from both patients studied

Parametera Patient 1 Patient 2

Ejaculate volume (ml)

pH

Viscosity

Spermatozoa count (×106/ml)

Normal spermatozoa (%)

Spermatozoa viability (%)

Spermatozoa immotility (%)

Biochemistryb

Citric acid (μmol/ejaculate)

Fructose (μmol/ejaculate)

a: Mean of 5 independent samples

b: One sample analysed

1.4

8.7

Normal

20.6

13.0

1.0

100.0

42.3

24.3

3.6

8.5

Normal

0.9

3.0

40.0

100.0

98.7

44.6

The ratio of sexual chromosomes was 1 1 as expected. The percentage of sperm DNA

damage was 52.6%, well above the normal range in the fertile population. The constitutional

karyotype was normal and no microdeletions were found on chromosome Y in whole blood

analysis. Apoptosis biomarkers were detected at higher levels in this patient and positive

A: thickening of the midpiece ( )

B: note the very evident thickening of the midpiece ( ); headless cell ( )

Figure 1 Morphological (A) and vitality (B) study of spermatozoa from patient 1

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controls than in negative controls by Western blotting analysis (Figure 3A). Microscopic fat

deposits (Red Oil O) or polysaccharides (PAS) were not observed. Papanicolaou staining

showed discrete thickening in the midpiece (images not shown).

Table 2 Aneuplody studies by FISH: probes for chromosomes 13, 18, 21, X and Y

Aneuploidy Total analysed cells Total altered cells Alteration rate (%) Above reference valueNullisomic

13

18

21

X/Y

Disomic

13

18

21

XX

XY

YY

1 346

2 462

1 346

2 462

1 346

2 462

1 346

2 462

2 462

2 462

4.4

4.5

4.8

3.5

0.7

0.8

0.4

0.8

1.5

0.2

59

112

64

37

10

20

6

20

37

5

Figure 2 Transmission electron microscopy (TEM) on spermatozoa from patient 1

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A: PARP1: Poly (ADP-ribose) polymerase. HF: positive control (human fibroblasts treated for 5 min with

ultraviolet light to induce apoptosis). There was an increase in apoptosis-related proteins (cleaved PARP1,

intact Caspase 3, and Annexin V) in the spermatozoa from patient 1

B: The β-tubulin is partially degraded in patient 2 compared with the pattern observed in controls. C1, C2, and

C3: sperm negative controls. P: Patient. Actin was used as a loading control

Figure 3 Western blotting for expression of apoptosis markers in the spermatozoa from patient 1 (A) and after

the addition of mouse monoclonal anti-β-tubulin antibodies on semen samples of patient 2 (B)

Patient 2

The light microscopy morphological (Figure 4A) and vitality (Figure 4B) studies showed

that 85% of spermatozoa had short flagella. Of those, 65% had thickened rigid flagella and

35% had rudimentary flagella which were not thickened. Only 3% were normal spermatozoa.

The unstructured and thickened fibrous sheath together with the hypoplastic and disorganized

tail midpiece (Figure 5A) were observed by TEM: the normal central pair of microtubules

was absent, leaving the structure known as the ‘9+0’ axoneme (Figure 5B,C), compared

Table 3 Diploidy studies by FISH: probes for chromosomes 13, 18, 21, X and Y

Diploidy Total analysed cells Total altered cells Alteration rate (%) Above reference valueAutosomic

chromosomes 13/21

Sexual chromosomes

1818XX

1818XY

1818YY

Total 18/X/Y

1 346

2 462

2 462

2 462

2 462

2.1

0.7

1.5

0.9

3.1

28

16

37

22

75

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Figure 4 Morphological (A) and vitality (B) study of spermatozoa from patient 2 showing coiled, rudimentary

and rigid/ thickened flagella

with the normal flagellar structure in the middle piece (Figure 6). Phase contrast and immu-

nofluorescence images of the fibrous sheath showed a hypertrophy of the fibrous sheath in

this patient’s samples (Figure 7). Western blotting for β-tubulin also revealed an abnormal

microtubular pattern in our patient versus the normal pattern of human sperm (Figure 3B). A

ciliary motility test on nose epithelium showed the two characteristic phases and the beat

frequency was 9 Hz (normal) and the cellular rotation test showed normal epithelial cell

rotation motion, thus a diagnosis of PCD was discarded.

Discussion

Here, we present two clinical cases of specific alterations in sperm morphology which

result in 100% immobility, and which are likely of a genetic origin: one with thickening of the

midpiece and a high rate of DNA fragmentation, and the other with loss of the central pair of

microtubules and fibrous sheath dysplasia. We found the low spermatozoa viability of the

sample from patient 1, the severe oligozoospermia in patient 2, and the full immotility of the

sperm from both patients particularly striking. Citrate and fructose levels in seminal plasma

were within the non-pathological range in both patients, which ruled out pathology of the

accessory sex glands (prostate and seminal glands).

Patient 1

Semen stains did not identify the nature of the abnormal deposit of material in the

midpiece so further investigation of the possible origin of this abnormal feature using different

methods was required. The apoptotic marker study was the most informative in this sense,

showing an increase in typical apoptotic proteins.

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Some studies have shown that sperm DNA damage is higher in ejaculated sperm

compared with testicular sperm, which reinforces the idea that sperm DNA damage

increases in transit from the testis to the epididymis and into the ejaculate[16]. At present we

do not know what determines a given cell’s apoptotic potential, and it is not clear whether the

apoptotic markers detected in spermatozoa are produced by apoptotic processes starting

before or after the ejaculation[17]. However, it is thought that endogenous caspases

(cysteinyl-aspartate-specific proteinases) cause apoptosis by inducing DNA fragmentation

during spermatogenesis.

A: Longitudinal section of a spermatozoa showing abnormal structure of the neck, middle, and main piece; the

head and the tail are disconnected in the midpiece ( )

B, C: Cross sections of the sperm flagellum showing disorganisation of the axonemal and periaxonemal components.

Lack of the central pair of microtubules and hyperplasic extension of the associated dense outer fibres

Figure 5 Transmission electron microscopy (TEM) on spermatozoa from patient 2

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Figure 6 Transverse section of normal tail axoneme

showing nine doublet microtubules, the

dynein arms, and radial spokes surround-

ing a central pair of singlet microtubules

This patient had almost absolute necrospermia, the aetiology of which is a poorly studied

cause of male infertility; hence we found very little literature which could help to explain the

pathology we have described. In this case, given that the patient had an infertile brother

(who could not be studied) we postulate that the necrozoospermia probably has a genetic

origin. The presence of stationary sperms, some with normal and others with abnormal

morphology, may indicate that they had undergone apoptotic changes. Furthermore, Burrello

et al.[18] suggest that genomic germ cell quality and the sperm remodelling that occurs during

Bottom row: immunofluorescence of the fibrous sheath. Labelling with mouse monoclonal anti-β-tubulin anti-

bodies shows the distribution of β-tubulin in the spermatozoa control, with a relatively weak green fluorescence

in the flagellum (a’), and a hypertrophy of the fibrous sheath an unusually high amount of β-tubulin in patient 2

(b’, c’) which results in the formation of short, “stump” defect, “stunted”, and coiled tails

Figure 7 Phase contrast images of semen control (a) and semen from patient 2 (b, c)

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spermatogenesis are not associated, i.e. a germ cell may be aneuploid or have its nucleus

altered by apoptosis, whereas a spermatozoa could have a normal morphology. The origin of

sperm DNA damage is multifactorial and can be attributed to internal and external testicular

factors: abnormal DNA packing during spermatogenesis, environmental toxins, oxidative

stress during sperm migration from the seminiferous tubules to the epididymis, DNA

fragmentation induced by endogenous caspases/endonucleases. This latter process occurs

during spermatogenesis and is caused by an error in the germ cell selection process by

Sertoli cells, thus allowing defective germ cells enter meiosis I and appear in the ejaculate, as

indicated by the expression of apoptotic spermatozoa[19]. Caspase activation plays an

important role in the initiation and activation of sequential apoptotic processes, causing the

cleavage of critical cellular substrates, including PARP1, a protein with molecular weight of

116 000 involved in DNA repair which can be cleaved by Caspase-3 in vivo[20]. PARP1

specifically binds at DNA strand breaks and helps to maintain cell viability and so its cleavage

facilitates cellular disassembly and serves as a cell apoptosis marker. Caspase-3 is activated

by cleavage, generating two subunits of 35 000 and 20 000 each. Annexin V, a protein with

molecular weight of 30 000, binds specifically to phosphatidylserine whose presence is also

related to apoptotic events[21]. Inactive Caspase-3 (35 000) was detected in our patient’s

sperm and in positive controls, but was absent in negative sperm controls. Active Caspase-

3 (20 000) was also detected in low quantities in the patient sperm but was not in negative

controls. Additionally, the presence of Annexin V was more pronounced in the patient than in

negative semen controls. Taken together, the presence of apoptosis-related proteins in this

patient’s sperm suggests an apoptotic origin for the abnormal deposits of material in the

midpiece. Moreover, the preservation of sperm morphology supports an apoptotic origin for

the changes observed in this case. Apoptosis, unlike necrosis, is characterised by conservation

of the cell structure.

Seminal FISH from the patient 1 showed abnormal meiotic recombination, presence of

aneuploidy (nullisomy) and diploidy, so a normal constitutional karyotype does not exclude

the presence of anomalies in sperm chromosomes; increase in the frequency of aneuploidy

in semen chromosomes of the patients with poor semen quality has been observed[22]. The

chromosomic study of the spermatozoa is of great interest and should be seriously considered

in infertile couples, mainly in the case of repeated in vitro fertilization failure.

Our patient had large numbers of thickened midpiece cells (85%), consistent with the

high rate of sperm DNA fragmentation observed (52.6%). Despite the necrozoospermia and

morphological alterations, his sperm did not present teratozoospermia: the change did not

affect the total sperm population and there was a high percentage of normal forms (13%).

This patient was remarkable for this singular accumulation of material in the midpiece of his

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sperm, which we observed in the vitality study but which was less evident in the morphological

and Papanicolaou stainings. The other cell elements were quite well preserved and the good

shape of the head was outstanding.

Proximal centriole abnormalities and degeneration of the midpiece, producing headless

sperm, has been described as ‘easily decapitated spermatozoa’ defect[23]. Head separation

appears to be the result of a specific morphogenetic process although a patient with head

separation from the tail in 100% of their spermatozoa has been described[24], although other

authors claim that loose heads are rarely found in semen[25]. The significant number of

detached heads found in our patient’s sample, associated with acephalic sperm, is also

interesting. We think that sperm head loss in this patient does not represent the extreme

phase of head-midpiece disjunction due to misalignment in neck area, but instead that the

deposition of abnormal material in the head-tail junctions makes the sperm unusually fragile

and frequently results in head separation. This is caused and enhanced by mechanical

phenomena when handling these particularly delicate samples in the laboratory.

Patient 2

In this case the total sperm immobility and short, rudimentary or rigid and thickened forms

of the flagellum drew our attention. Anomalies present in the samples included the absence

of central pair doublets and the presence of coiled, short/stump tails but not all the spermatozoa

were affected (85%). This incomplete form of flagellar pathologies has already been

described by other authors[26,27].

Given the morphological findings described above, and the clinical history (previous

surgical intervention on the tympanum), we chose to first check the diagnosis of PCD.

Expression of axonemal alterations in sperm tails and respiratory cilia is variable: Chemes

et al.[28] described two cases of concomitant ciliary dyskinesia and hyperplasia of the fibrous

sheath; Neugebauer et al.[13] reported a patient with 100% immotile spermatozoa in which the

central pair of microtubules was missing, a change which was also reflected in 15% of nasal

cilia. This defect, called the ‘9+0’ axoneme, is associated with a thickened fibrous sheath.

As Armengot et al.[15] pointed out, video analysis is probably more useful than studying

the ultrastructure of cilia when attempting to screen for PCD. Escudier et al.[29] found a

normal nasal cilia beat frequency in 84 % of infertile men (11/13), while significant ciliary

ultrastructural abnormalities were observed in the nasal cilia of 92% of infertile male patients

(12/13), with only one patient in the cohort also suffering severe respiratory disease. However,

PCD can only be definitively rejected by findings related to ciliary function, because 30% of

PCD patients show normal ultrastructural respiratory cilia[30]. There are also reported cases

of discordance between flagella and cilia: immotile sperm with ultrastructural axoneme

alterations but normal cilia (motility and morphology) and vice versa[9,31]. The nasal cilia

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function in our patient showed a normal ciliary beat frequency and pattern, and no clinical

significance in terms of pulmonary involvement was found. Although he had repeated ear

infections that required a surgery, together these clinical findings ruled out PCD.

The sperm head-tail attachment in our patient was normal and was not associated with

acephalic sperm, unlike observations from similar cases[4,32]. Patients with totally immotile

spermatozoa due to ‘9+0’ ultrastructural flagellar anomalies have been associated with

polycystic kidney disease in approximately 25% of cases[33], although we did not find such an

association in our patient. In this case, the lack of central microtubules and hyperplasic

extension of the dense outer fibres suggests that the normal fibrous sheath structure may

help to maintain the integrity of the axonemal microtubules[8]. The flagella morphological

findings were compatible with DFS.

Chemes et al. suggested a genetic origin for this DFS[25]. Several human flagellar

defects have been found in brothers of consanguineous parents, such as the absence of

dynein arms, detached tail, ‘9+0’ flagella syndrome, DFS, and short/stump tail, indicating a

genetic basis for this particular fibrous sheath anomaly[34]. Most sperm anomalies with a

genetic basis are determined by recessive autosomal mutations, resulting in abnormal or the

complete lack of motility[32], which unfortunately can be transmitted by assisted reproduction

techniques[12,35]. In our patient’s case, follow up studies following the patient’s genealogical

family tree could help to identify inheritance patterns which might associate the abnormal

morphology with a genetic cause.

Conclusions

As discussed in this paper, the high level of DNA fragmentation and the presence of

apoptosis-related proteins in sperm from patient 1 suggest an apoptotic origin for the abnormal

deposits of material in the midpiece. On the other hand, the ultrastructural study of the sperm

tail showing the absence of the central pair of microtubules together with the normal nasal

ciliary test performed in patient 2 ruled out PCD, being these findings consistent with DFS.

These features cannot be identified with a basic spermiogram nor with routine biochemical

analysis, and so promoting close collaboration between clinical andrology laboratories and

biological research centres in order to achieve better diagnoses of these alterations is essential.

Acknowledgement

We thank María Ciria Calduch and Mario Soriano Navarro for their assistance with the

immunocytochemical and ultrastructural studies.

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(Received on April 22, 2014)