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The aim was to examine the morphology of spermatozoa with different staining methods and aimed to find the better staining methods for morphology of spermatozoa in our study. Randomized 67 patients taken for the study who were admitted to Assisted Reproductive Techniques Unit. In the first part of the study, smears were stained with Hematoxylin Eosin (HE), Toluidin Blue (TB), Giemsa, Wright, ferrous Weigert haematoxylin stain, Orange G, eosin-aniline blue dye, Shorr Method, Papanicolau, Berg Method, Light Green stain, Acridine Orange (AO) and Janus Green dyes. In the second part of the study, smear preparations of 10 patients with normozoospermic were stained with HE, Toluidin Blue (TB), Shorr Method and Papanicolau. Four measurements were made including the middle piece, head length- head width and tail length for 200 spermatozoa with normal morphology. Comparisons were made between the stains that which showed a better morphology. Condensation assessment was not possible in smears stained with Shorr, Berg, Method, AO. Better assessment of condensation could be made in other stains. In the second part the smallest values belonged to of TB stain according to measurements of head of the spermatozoa. There was a significant difference at the head length with TB stain. Although measurements of Shorr and Papanicolau are close to each other and the largest values belonged to Papanicolau dye. It was concluded that measurement values in human sperm morphology could alter with the used staining method.
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Int. J. Morphol.,
30(4):1544-1550, 2012.
Assessment of Spermatozoa Morphology under Light
Microscopy with Different Histologic Stains and
Comparison of Morphometric Measurements
Evaluación de la Morfología de los Espermatozoides bajo Microscopía Óptica con
Diferentes Tinciones Histológicas y Comparación de las Mediciones Morfométricas
*
Emine Aksoy;
**
Tahsin Murad Aktan;
**
Selcuk Duman &
**
Gokhan Cuce
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica
con diferentes tinciones histológicas y comparación de las mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
SUMMARY: The aim was to examine the morphology of spermatozoa with different staining methods and aimed to find the
better staining methods for morphology of spermatozoa in our study. Randomized 67 patients taken for the study who were admitted to
Assisted Reproductive Techniques Unit. In the first part of the study, smears were stained with Hematoxylin Eosin (HE), Toluidin Blue
(TB), Giemsa, Wright, ferrous Weigert haematoxylin stain, Orange G, eosin-aniline blue dye, Shorr Method, Papanicolau, Berg Method,
Light Green stain, Acridine Orange (AO) and Janus Green dyes. In the second part of the study, smear preparations of 10 patients with
normozoospermic were stained with HE, Toluidin Blue (TB), Shorr Method and Papanicolau. Four measurements were made including
the middle piece, head length- head width and tail length for 200 spermatozoa with normal morphology. Comparisons were made
between the stains that which showed a better morphology. Condensation assessment was not possible in smears stained with Shorr,
Berg, Method, AO. Better assessment of condensation could be made in other stains. In the second part the smallest values belonged to
of TB stain according to measurements of head of the spermatozoa. There was a significant difference at the head length with TB stain.
Although measurements of Shorr and Papanicolau are close to each other and the largest values belonged to Papanicolau dye. It was
concluded that measurement values in human sperm morphology could alter with the used staining method.
KEY WORDS: Spermatozoa; Morphology; Histological stains.
INTRODUCTION
Approximately 15 % of married couples have
infertility problems. Twenty percent of them originate from
men, 30-40 % originate from both men and women. So that
half of the infertility couples have male-factor infertility
(Spira 1986; Anafarta et al., 2007). ICS (intracytoplasmic
sperm injection) was first performed by Palermo et al. (1992),
many of the infertile couples could not be treated before,
have been treated by ICSI.
Morphology of spermatozoa is an important for a
successful fertilization and early embryonic development
in assisted reproductive techniques (Kuster et al., 2004).
Final Tygerberg Classification Criteria described by Kruger
in 1986 and the WHO classification are the most important
spermatozoa morphological classifications. According to
Kruger's sperm, the head, neck and tail sections should be
normal, the boundaries of the head should be smooth and
oval and 70% of head should be coated by acrosome. The
head of the sperm head length should be 4-5 µm and must
have a width of 2.5-3.5 µm. The length of the tail should be
an average of 45 µm. The middle part must be thin and long
and width should be less than 1 mm, length should be of 1.5
times the length of the head. The tail should be thinner than
middle piece, should be flat and not wrinkled, should not
contain broken parts and cytoplasmic debris. In addition,
the sperm should not have the neck, middle piece, tail
abnormalities and a large cytoplasmic droplet more than half
of the spermatozoon head in the neck area (WHO, 1999;
Mehta et al., 2006).
The sperm must be stained for a better assessment of
morphology. As there are many staining methods as
*
Department of IVF, Central Laboratory of Education and Research Hospital, Konya, Turkey.
**
University of Selcuk, Faculty of Meram Medicine, Deparment of Histology and Embryology, Konya, Turkey.
1545
Papanicolaou, haematoxylin, eosin, Giemsa, Diff-Quick for
sperm staining (Sanchez et al., 1994). Morphological features
of spermatozoa are expressed in numerical values.
Methods used in the staining may cause a slight change in
the measurement values of spermatozoa because fixatives
can cause cells to shrink a little. For example, 3-5 µm length
and 2-3 µm wide measurements are considered normal for
the head of spermatozoa in the method of Papanicolaou.
These values change 5-6 and 2.5 µm - 3.5 µm in the Diff-
Quick staining method. In both the middle piece should be
1 µm thick, the length of this value should be up to 1.5 ti-
mes of this value. The tail section should be 45 µm
length tapering gradually (Irez et al., 2007). Because of these
differences, we examined morphology of spermatozoa with
different staining methods and aimed to find the better
staining methods for laboratories in our study.
MATERIAL AND METHOD
In this study randomized 67 semen samples were
obtained from patients for the study which were admitted to
University of Selcuk, Faculty of Meram Medicine, Assisted
Reproductive Techniques Unit. These patients have
teratozoospermia (n=25), Oligospermia (n=20) and
normozoospermia (n=22).
Smear preparations were prepared from washed
samples; the smears were dried at room temperature and
fixed in methyl alcohol. In the first part of the study, smears
were stained with (HE), TB, Giemsa, Wright, ferrous Weigert
haematoxylin stain, Orange G, Eosin-aniline blue dye, Shorr
Method, Papanicolau, Berg Method, Light Green stain,
Acridine Orange (AO) and Janus Green dyes. Preparations
were evaluated under the light microscope.
In the second part of the study, smear preparations of
10 patients with normozoospermic were stained with HE,
Toluidin Blue, Shorr Method and Papanicolau. Evaluation
of the sperm was based on Kruger strict criteria. Four
measurements were done including the middle piece, head
length- head width and tail length for 200 spermatozoa with
normal morphology. Comparisons were made between the
stains that which showed a better morphology and which is
more appropriate for Kruger strict criteria by the statistical
analysis. Analysis of the normal distribution of data was
done.
Statistical comparisons between groups were
performed with one-way analysis of variance (ANOVA)
(P<0.05). Analysis of the normal distribution of data was
examined with Kolmogorov-Smirnov Test.
RESULTS
Head, middle piece and tails of spermatozoa were
seen clearly with HE. Acrosome core rate was extremely
good in the form of light and dark purple (condensation).
The stain was homogeneously distributed on the head and
dark purple color was seen. The middle piece and tail were
seen clearly and can be evaluated very well (Fig. 1). The
ideal image was obtained by staining spermatozoa with 0.1%
TB. The condensation of the head can be chosen by the
whitish blue color. Condensation and morphology of the head
can be selected clearly (Fig. 2).
Fig. 1. Spermatozoa stained with H-E (FMBx 330).
Fig. 2. Spermatozoa stained with TB.
The spermatozoa were stained dark blue purple with
Giemsa. Head morphology and condensation could be seen
very well but the middle piece and the tail did not seen clear.
Spermatozoa were painted in shades of blue by Lilly Wright
dye. Condensation in the head and morphology of
spermatozoa were well-selectable. In an unexpected finding
it was observed that some of spermatozoa including head
damage, stained pink in the head. Spermatozoa heads were
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica con diferentes tinciones histológicas y comparación de las
mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
1546
painted in black and navy blue with Weigert dye with ferrous
H. Spermatozoa can be selected properly. Condensation
could be distinguished on the head of spermatozoa and the
head had been stained a dark purple color. Middle part and
the tails were stained pink. Middle part was stained more
than the other dyes. Flagellas are easily evaluated.
Morphology of spermatozoa was clear and smooth with
Orange G. Condensation could be seen in the head of the
spermatozoa and spermatozoa stained fully orange. Flagella’s
can be selected easily (Fig. 4).
flagella’s stained green. The middle piece was pronounced.
Flagella’s were painted pale but was selectable (Fig. 6). The
cell membrane stained a darker color than cytoplasm with
Shorr that is why the morphology of spermatozoa and the
cell borders could be seen very significant. The head is
stained as a dark yellow color homogeneously. Condensation
assessment could not be done but the middle part and flagella
can be evaluated as very clear Therefore, all
the morphological defects of spermatozoa could be easily
evaluated (Fig. 7).
Fig. 3. Spermatozoa stained with Weigert’s ferrous Haematoxylin.
Fig. 4. Spermatozoa stained with Orange G.
Head of the spermatozoa stained light-dark-blue with
Eosin-Aniline blue. Head morphology and the condensation
were seen extremely well. Middle part and the flagella’s can
be seen well, but it was pale blue. The heads of spermatozoa
stained yellowish brown with Papanicalou dye and
condensation could be seen. Middle part and the flagella’s
were clearly stained dark brown (Fig. 5).
Spermatozoa morphology can seen well with light
green dye. Condensation could be seen clearly and head of
the spermatozoa stained green-blue. Middle parts and the
The heads of spermatozoa were stained pale pink-lilac
color with Berg Method. Morphology of spermatozoa was clear,
but condensation of head could not be evaluated. Morphology
of the spermatozoa was clear but condensation at the head was
not evaluated with AO stain. When AO stain applied with
haematoxylin, haematoxylin only stained the membrane around
nucleus and spermatozoa observed like Y letter.
Morphology of spermatozoa with the supra vital dye
Janus Green was clear, especially the middle piece (Fig. 8).
Additionally, preparations of 10 normozoospermia patients
Fig. 5. Spermatozoa stained with Papanicolau.
Fig. 6. Spermatozoa stained with Light Green.
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica con diferentes tinciones histológicas y comparación de las
mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
1547
stained with H-E, TB, Shorr and Papanicolau were assessed
according to Krugers criteria. Spermatozoa were measured in
terms of 4 parameters as head length, head width, middle part
length and tail length. Statistical analysis were made using one-
way variance analysis (ANOVA) and Post hoc Tukey HSD
test and which stain showed better spermatozoa morphology
and which was more appropriate for Krugers strict criteria
were determined. The smallest values belonged to TB
according to measurements of head of the spermatozoa. There
was a significant difference at the head length with TB stain.
Although measurements of Shorr and Papanicolau are close to
each other and the largest values belonged to Papanicolau stain
(Table I) (P<0.028).
DISCUSSION
Assessment of spermatozoa morphology is an
important parameter in the diagnosis of infertile men. Due
to the use of increasing amounts of IVF (in vitro fertilization)
techniques, studies are also focused on the morphology
of spermatozoa about the role in fertilization. Evaluation
can be done with staining spermatozoa with a variety of
methods from the fresh semen by electron, fast-contrast and
light microscope. There are many staining methods to
evaluate the morphology of spermatozoa (Uven et al., 1998).
García-Herreros et al. (2006) used Hemaklor, Harris
haematoxylin and PanOptic fast staining techniques to make
standardization of the sampling methods, sample preparation
and staining for the head of the spermatozoa with computer-
assisted morphometric analysis (CASMA). They concluded
that Haematoxylin and Hemaklorun are the best staining
methods for evaluation of sperm heads.
Soler et al. (2005) compared 3 different dye methods
for assessment of spermatozoa morphometry with
computerized Integrated Semen Analysis System (ISAS).
They evaluated 200 spermatozoa cells stained with
Hemacolor, Diff-Quik and harris haematoxylin. While they
found all techniques were also useful in that used in staining
of spermatozoa but Diff-Quick stain showed a significant
difference.
TB is used in the evaluation of the nuclear chromatin
of spermatozoa by identifying the absence or rupture of
disulfide bonds. Beletti & Mello (2004) examined
relationship between spermatozoa morphology and
spermatozoa chromatin condensation with TB and Feulgen
reaction. TB is a effective stain for the evaluation of changes
in spermatozoa chromatin. Our study is compatible with the
knowledge of the literature. TB dye staining method is a
simple method and allow simultaneous evaluation for
morphology and condensation of spermatozoa. TB is an ideal
dye for routine use in this area.
Chromatin condensation abnormalities of
spermatozoas can be observed with Aniline Blue. The
technique can be used to assess quality of spermatozoa (7).
Avcı (2006) used different fixation protocols to the semen
samples and stained with Aniline Blue, Acridine Orange and
Chromamycine A3 (CMA 3). The result for the quality of
staining did not differ between the dyes. Hammadeh et al.
(2001) aimed to identify the spermatozoa chromatin
condensation values for assessment of male fertility. 165
semen samples had taken from 90 patients and 75 healthy
individuals, stained with Aniline blue. They examined some
Fig. 7. Spermatozoa stained with Shorr.
Fig. 8. Spermatozoa stained with Janus Green.
Table I. Measured values of head length and width in
spermatozoa stained with HE, TB, Shorr and Papanicolau.
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica con diferentes tinciones histológicas y comparación de las
mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
Length WidthGroup
Mean ±SS Mean ±SS
H-E 4.75±0.15 2.6±0.16
TB 4.59±0.25 2.56±0.04
Shorr 4.86±0.15 2.63±0.08
Papanicolau 4.89±0.31* 2.69±0.25
P 0.028 0.314
1548
classical semen parameters like the chromatin condensation,
spermatozoa morphology, motility and progressive motility.
They claimed that there was not a correlation between the
chromatin condensation and the others. It was recommended
that chromatin condense must be included, in routine
laboratory observations.
Morel et al. (1998) randomly selected 47 patients
applied for semen analysis, they examined the diversity of
common morphological d
isorders in the human spermatozoa
among individuals. In order to show their connection with
the nuclear maturity, sperms stained with Aniline Blue,
Spermatozoa morphology defects differences seen between
individuals and they found a significant relationship
between frequent of defects and degree of nuclear maturate.
Our study is compatible with the knowledge of the
literature. Aniline Blue dye provides the ideal image to
measure the rate of acrosome head. Condensation and
morphology can be assessed together.
Tartaglione & Ritta (2004) examined the prognostic
spermatological values of frozen dissolved spermatozoa to
estimate in vitro fertility. Estimating plasma membrane and
acrosome integrity are important values for normal
functions of spermatozoa, for this aim, they examined
smears stained with Trypan Blue and Giemsa. They
concluded that these stains can be used for the prognosis
of the fertility in the semen used for IVF.
Our evaluation was consistent with the literature of
Giemsa and Wright stains. Condensation and head
morphology of spermatozoa were well-selectable. The
middle piece and tail could be seen. An unexpected finding
that tail and head completely stained pink in the some of
the spermatozoa which had damaged head morphology. A
further study was suggested to explain the reason.
Spermatozoa morphology and condensation were clear and
smooth with Orange G stain. But staining process was too
long that’s why Orange G stain had no superiority.
Shorr technique is preferred in many laboratories
due to the insufficient relationship in the results of IVF.
Henkel et al. (2005) evaluated decreased motility of
spermatozoa in elderly men with Shorr technique. The
percentage of stained spermatozoa with normal and
abnormal flagella’s was evaluated and found a negative
correlation between the stained abnormal flagella’s and
speed ratio and motility of the spermatozoa in elderly man.
Milingos et al. (1996) examined the sperm from 89
infertile couple and stained aniline blue and Shorr. They
claimed that Shorr dye showed a better significant
morphology than aniline blue. Our study is compatible with
the knowledge of the literature. Shorr is a suitable dye for
morphologic evaluation of spermatozoa but it is not
considered to be appropriate for the assessment of
condensation.
Papanicolaou's method allows assessment of
morphological of healthy human spermatozoa and also
allows the separation of immature germ cells. Menkveld et
al. (1997) examined the relationship between human
spermatozoa morphology and function of acrosome.
Acrosome reaction evaluated at normal, small and large
size in living and dead spermatozoa with triple stain.
Spermatozoa with small acrosome were physiologically
more susceptible to cell death and loss of acrosome. In
addition, acrosome size reflects functional capacity of
spermatozoa, thus the potential of male fertility can be
evaluated.
Spermatozoa stained with Orange Acridine were
very pale and could not be evaluated. When stained with
AO+ Haematoxylin, only the membrane around the nucleus
stained with haematoxylin and then spermatozoa was seen
in the form of the letter Y. New researches are recommended
to investigate whether this image has a clinical value.
Michael et al. (1987) stained and studied on spermatozoa
and immature germ cells by using phase contrast
microscope. With janus green B and tymol; head of
spermatozoa, nuclear membrane, middle section was
stained light green but nucleus was stained dark purple. In
this method, janus green provided an excellent staining for
evaluation of spermatozoa morphology.
In our study, spermatozoa heads appeared as whitish-
bluish light pale coloured, middle sections and tails were
stained dark blue. Our study was appeared to be compati-
ble with the literature and as the result of our study.
Spermatozoa middle section which is a rich section for
mitochondria’s were revealed clearly and was displayed
once again by janus green.
At second phase of our study, samples of 10 patients
with normazoospermatozoa stained with HE, TB, Shorr and
papanicolau and analysed by Kruger strict critters. Four
different measurement
s including head length, head width,
the mild section length and the tale length were done. We’ve
had different results than literature data. Gago et al. (1998)
used computer assisted image analysis systems with different
staining methods for morphological evaluation of
spermatozoa heads. They have found different results for
spermatozoa head measurement parameters with
Haematoxylein, diff-quick (Giemsa-Wright) and Hemaclor
stain each. According to this results; it is concluded that
spermatozoa head measurements are affected significantly
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica con diferentes tinciones histológicas y comparación de las
mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
1549
by fixation procedures and staining techniques. When we
compared haematoxylin and Giemsa-Wrihgt stains as
morphologically we could not find a significant difference.
Our study was not compatible with the literature in this sense.
Root Kustritz et al. (1998) investigated spermatozoa
morphological anomaly types and their percentages by using
Giemsa-Wright stain, Eosin Y/nigrosin and Eosin B/
nigrosinstains with light microscope. In addition unstained
samples were evaluated by using phase contrast microscope.
They found higher percentile average of spermatozoa
morphologies stained by Diff-Quick as a result of their study
and concluded that preparation and staining techniques can
change the evaluation of spermatozoa morphology.
Menkveld et al. (2003) examined different staining
and washing procedure’s affects spermatozoa morphology
by using manual and computerised methods. They used
Papanicolau and Diff-Quick staining methods at 20 semen
samples. Manual assessments gave better results than
computer assessments. Also spermatozoa morphological
evaluations were better with once washed Diff-Quick stained
smears than Papanicolau stained samples. In our study;
morphological assessments were better with Papanicolau
stain than Giemsa and Wright stain. In this sense our study
was not compatible with literature data.
In our study morphometrical measurements of
spermatozoa heads were smallest with TB stained samples.
Shorr and Papanicolau stains have close measurements to
each other but the maximal values belonged to Papanicolau
stain. In the end we can say “for morphological assessment
of spermatozoa, our special stains HE, TB, Shorr and
Papanicolau are the best dyes as staining quality”. Although
many studies in literature are related to our work, there are
only limited numbers of studies done for spermatozoa
morphological assessment with comparing results of
different stains. When it is thought the fact that having baby
with reproduction techniques assists male infertility; the
studies for morphological evaluations of spermatozoa’s still
remain important to select high quality spermatozoa’s.
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Assessment of spermatozoa morphology under light microscopy with different
histologic stains and comparison of morphometric measurements.
Int. J. Morphol., 30(4):1544-1550, 2012.
RESUMEN: El objetivo fue examinar la morfología de los espermatozoides con diferentes métodos de tinción y encontrar los
mejores métodos para su estudio. Fueron seleccionados para el estudio, de manera aleatoria 67 pacientes, quienes ingresaron a la Unidad
de técnicas de reproducción asistida. En la primera parte del estudio, se realizaron y tiñeron frotis con hematoxilina eosina (HE), Azul de
Toluidina (AT), Giemsa, tinción de Wright, Hematoxilina Férrica de Weigert, Anaranjado G, tinción eosina-anilina, método de Shorr,
Papanicolau, método Berg, tinción verde brillante, anaranjado de acridina (AO) y tinción verde Janus. En la segunda parte del estudio, se
realizaron frotis de 10 pacientes con normozoospérmicos y se tiñeron con HE, AT, Método Shorr y Papanicolau. Se realizaron cuatro
mediciones: ancho de la cabeza, longitud de la cabeza, parte media y cola, sobre 200 espermatozoides con morfología normal. Se
compararon las tinciones que mostraban mejor la morfología. La evaluación de la compactación no fue posible en los frotis teñidos con
los métodos de Shorr, Berg y AO. Una mejor evaluación de la copactación podría hacerse en otras tinciones. En la segunda parte los
valores menores correspondieron a la tinción de AT en relación a la medición de la cabeza de los espermatozoides. Hubo una diferencia
significativa en la longitud de la cabeza con tinción de AT. Las mediciones en los frotis con técnicas de Shorr y Papanicolau fueron
similares, con valores más altos bajo tinción de Papanicolau. Se concluyó que los valores de la medición morfológica en espermatozoides
humanos podrían ser alterados según el método de tinción utilizado.
PALABRAS CLAVE: Espermatozoides; Morfología; Tinciones histológicas.
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Correspondence to:
Emine Aksoy
Department of IVF
Central Laboratory of Education and Research Hospital,
Konya
TURKEY
Email: draksoyemine@hotmail.com
Received: 19-11-2011
Accepted: 22-05-2012
AKSOY, E.; AKTAN, T. M.; DUMAN, S. & CUCE, G. Evaluación de la morfología de los espermatozoides bajo microscopía óptica con diferentes tinciones histológicas y comparación de las
mediciones morfométricas.
Int. J. Morphol., 30(4):1544-1550, 2012.
... Some authors recommend the use of combination of stains to overcome this limitation for morphometric measurements. 3,4 The process of slide smear preparation from the semen sample is time consuming. Assessment of sperm morphology requires semen smear preparations to be of high quality. ...
... Sperm quality is important for successful fertilization and early embryonic development in assisted reproductive techniques. 4 It is considered to be one of the best discriminators for fertilization potential. 3 Until the 20th century, little attention was given to assessment of sperm morphology. ...
... Morphology of sperms can be assessed better when they are stained and Papanicolaou stain gives good staining of spermatozoa. 3,4 Aksoy E et al. 4 used different staining methods to assess morphometric measurements and morphology of spermatozoa under light microscopy, in 67 patients. They found changes in the morphometric values of spermatozoa because the fixatives induced slight cell shrinkage. ...
Article
BACKGROUND Semen analysis is an integral part of work up for infertility in men, with sperm morphology being an important qualitative parameter. Qualitative defects can affect any part of the sperm and are classified as defects in the head, middle piece, and tail, based on morphology. The focus of the study was to assess qualitative defects in sperms by light microscopy, in semen with normal sperm counts. METHODS This study is hospital based, descriptive, retrospective study. Of the semen samples received in the clinical laboratory, fifty with normal sperm counts were included in the study and processed according to standard protocol. For evaluation of qualitative defects by sperm morphology, smears were fixed in ethanol, stained with Papanicolaou stain [PAP], and assessed under light microscope. RESULTS The 50 semen samples included in the study had sperm counts ranging from 15 to 80 million / ml. Thirty samples had less than 10 % abnormal forms, fourteen samples had 11 - 20 % abnormal forms, five samples had 21 - 30 % abnormal forms and one sample had 40 % abnormal sperms. Qualitative defects were classified as morphological abnormalities in head, neck, and tail. Of the fifty cases, most defects were found in the head, followed by those in the neck and tail. Common defects noted were double head (44 %), abnormal sized heads, and bent neck (48 %). Coiling was a common defect noted in the tail (10 %). Most sperms showed a combination of defects. CONCLUSIONS Qualitative defects in sperm morphology are often seen in samples with normal sperm counts. Assessment of microscopic characteristics of human spermatozoa is as important as count and motility in the complete evaluation and work-up of semen samples in cases of infertility. KEY WORDS Semen, Sperm, Quality, Microscopy, Morphology
... Se puede realizar utilizando diferentes métodos de preparación incluyendo una tinción de fondo tal como eosina-nigrosina (EN), coloraciones más específicas como papanicolau o giemsa, o una técnica de montaje húmeda en la que la muestra de semen se fija en una solución salina de formol tamponada (Aksoy et al., 2012). Se deben evaluar por lo menos 100-200 espermatozoides y determinar la incidencia de defectos morfológicos. ...
Thesis
Full-text available
Artificial insemination (AI) is an assisted reproductive technique that allows for both genetic progress and more efficient stall management. This consists of obtaining semen from a stallion, which is deposited on the female reproductive tract by means of an insemination pipette. AI with refrigerated semen has had an important impact on equine reproduction especially in the last decades. The semen to be cooled is diluted in specific media that allow it to tolerate the temperature drop. Although the diluents have the capacity to allow the cooling of the semen and in this way to maintain the fertilizing capacity of the spermatozoa, they only do it for a limited time. This is because, during refrigeration, changes in the permeability of the cell membrane that allow the ingress of ions into the spermatozoid, among them calcium (Ca ++), occur. This phenomenon can trigger premature training, accelerating the acrosomal reaction and leading to cell death. The most commonly used diluents are based on the formula described by Kenney, compounds based on skim milk powder and a sugar. Its use was extended by its simplicity in the elaboration and low cost. However, the high content of Ca ++, contributed by milk, would favor the premature triggering of training. Under natural conditions, the training must take place near the site of fertilization, in the female reproductive tract, and is indispensable for the spermatozoon to be able to fertilize the oocyte. If it is triggered anteriorly and out of the reproductive tract, the sperm loses its fertilizing capacity and accelerates its death. We propose that the addition of ethylenediaminetetraacetic acid (EDTA), a chelating agent, to diluents based on skim milk powder improves the quality of equine semen, prolonging its half-life. The decrease of free Ca ++ due to the chelating effect possibly decreases the number of spermatozoa trained prematurely. Two diluents were tested: a Kenney (K) -type diluent plus 2.5 mM EDTA, and a diluent K plus 5 mM EDTA. The samples were refrigerated at 5 ° C and vigor, movement mobility, survival and percentage of capacitated, uncapacitated and acrosomal-reactive cells were evaluated at 0, 24, 48 and 72 hours post-refrigeration. A beneficial effect of K 2.5 mM EDTA diluent was observed on all parameters studied, maintaining the sperm quality for 48-72hs of cooling. In confirmation of the proposed hypothesis, the addition of 2.5 mM EDTA to the Kenney diluent further maintains spermatozoa in a non-capacitated state during refrigeration at 5 ° C. In conclusion, the addition of 2.5 mM EDTA improves the functionality of the Kenney diluent, maintaining the fertilizing capacity of the refrigerated spermatozoa at 5 ° C. This improvement would allow artificial insemination 48 hs. post-cooling the semen or even later, although further studies are needed to confirm this hypothesis.
... Different types of sperms i. e. normal, big head, pin head, amorphous head, banana shaped etc. were observed. Total 1500 number of sperm heads of each treatment was analyzed [17,28,29]. ...
Research
Full-text available
The genotoxicity of the ethanolic extract of betel nut was evaluated using sarcoma 180 tumour bearing mouse considering sperm motility, sperm viability, biochemical estimation of fructose in seminal fluid and sperm head morphology assays. Sperm head morphology was studied by HE staining and Toluidine blue staining method. But Toluidine blue staining method is a reliable method to evaluate the DNA damage of sperms. Ethanolic BNE (betel nut extract) can suppress the percentage of sperm motility, sperm viability and seminal fructose level. In addition, it can also enhance the percentage of DNA damaged sperms. Moreover, histological sections of testes have been studied in control and BNE treated sarcoma 180 tumour bearing mice to highlight the potential toxic effect of BNE. The significant decreasing rate of seminal fructose concentration, sperm motility as well as viability and increasing rate of sperm head abnormality in different doses of treated series may be as a result of different toxic alkaloid ingredients present in BNE. Therefore, the results showed the potential of the BNE to induce different types of germ cell abnormalities in tumour bearing male mice.
... Although the direct morphological examination was carried out on the selection of spermatozoa for ICSI, until now morphological criteria for spermatozoa have not been applied in semen analysis. 11 Our study is the first study which compared the Lenshooke TM SQA X1 Pro with standard WHO method. Another study used Makler chamber for concentration and motility assessment in manual method. ...
Research
Background: Lenshooke TM Semen Quality Analyzer (SQA) X1 Pro is an automated semen analysis. The accuracy of Lenshooke TM SQA X1 Pro has never been analyzed with World Health Organization (WHO) standard method. Aim: This study aims to examine whether the Lenshooke TM SQA X1 Pro method provides reliable results according to the WHO standard method. Methods: This study was a laboratory analytic observational study using 60 patients in Andrology clinic of Dr. Soetomo Hospital. The concentration, progressive motility (PR), total motile sperm count (TMSC), and morphology results of the Lenshooke TM SQA X1 Pro and standard method were analyzed statistically using correlation, Bland Altman, and diagnostic test. Results: Significant correlation between two methods were found in all parameters (concentration: r = 0,970; PR: r = 0,781; TMSC: r = 0,952; morphology: r = 0,568). The mean difference for concentration, PR, TMSC, and morphology between the two examination methods were 1,165 million/ml, 7,05%, 7,584 million/ejaculate, and 2,25%. However, it found that the correlation and agreement were weaker in sample with low number of spermatozoa per high power field. The results revealed a sensitivity of 100%, 81%, and 59% for oligozoospermia, astenozoospermia, and teratozoospermia, respectively. The specificities were shown to be 100%, 74%, and 100% for oligozoospermia, astenozoospermia, and teratozoospermia, respectively. Conclusion: The Lenshooke TM SQA X1 Pro gives a reliable result for determining oligozoospermia and asthenozoospermia, but in the situation that the clinicians need the accurate data, standard method should be used.
... Sample processing for embedding in paraffin was performed according to Soliman and Abd-Elhafeez (2016). The protocols for histochemical staining using H&E, safranin O, methylene blue, Mallory trichrome, and PAS were performed following Abd-Elhafeez and , and orange G staining was performed according to Aksoy, Aktan, Duman et al. (2012) and Krishna and Kumar (2016). All stained sections were imaged using a Leitz Dialux 20 Microscope and a Canon digital camera (Canon PowerShot A95). ...
Article
The current study investigated the macroscopic and microscopic differences between pennaceous and plumulaceous feathers. The morphology of the barbules distinguished pennaceous and plumulaceous feathers, particularly the shape of barbules during their development. In pennaceous feathers, the initial barbules were large and elongated or pyriform in shape, while plumulaceous feathers had small, thin, elongated initial barbules. The spinous barbules were characteristic of pennaceous feathers. The histochemical reactivity of both feather types for Mallory trichrome, orange G, and acridine orange, safranin O, PAS, and methylene blue was determined. Keratin was detected by Mallory trichrome, orange G and acridine orange. In conclusion, the histochemical properties of pennaceous and plumulaceous feathers of quail, particularly the distribution and nature of keratin during development, should be considered in future studies. The unique morphological features of pennaceous and plumulaceous feathers could be used as a guide for phylogenetic identification. This article is protected by copyright. All rights reserved.
... Each reagent used in staining methods or fixative types can cause either the sperm cell to swell or shrink by penetrating its The effect of the staining technique on morphological and morphometric parameters of boar sperm membrane and influencing the osmotic balance. [4,[76][77][78][79][80] Procedures involving higher numbers of stages and chemicals are more likely to damage the sperm cell, resulting in changes in its dimensions. PAP staining involves the use of over 12 different chemicals, some of which can cause extreme hypoosmotic conditions, and thus shrinking the spermatozoa of different species including humans. ...
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Full-text available
Sperm morphology and morphometry are important parameters in predicting fertility. Sperm are considered to be normal if the shape and size of the head, midpiece and tail fall within the classification for a given species. It is important to select the appropriate technique for staining the semen of a given species, because, as many authors have pointed out, some methods work well for one species but are not suitable for analysing another. The aim of the study was to assess the morphometric parameters of boar sperm following the use of different staining techniques and to verify the hypothesis that the staining technique affects the morphometric parameters of sperm. The staining method was found to significantly affect the dimensions of the boar sperm head. The semen stained by the SpermBlue technique had the closest morphometric sperm head parameters to those of the unstained sperm, so this technique, rather than the routinely used eosin and gentian complex, should be the leading technique in the evaluation of boar sperm morphometry. Silver nitrate staining reveals the structure of the sperm in the most detail; this method can be considered universal, and can be used independently or to supplement routine diagnostics. As the staining technique should interfere as little as possible with the structure of the sperm, while revealing its morphology in as much detail as possible, it is crucial to establish the natural dimensions of the unstained sperm head before determining the optimal technique and its reference values. The recommended or most commonly-used techniques are not always the best options for the staining and analysis of sperm of a given species.
... For the analysis of sperm motility, the computer-assisted semen analysis system was used. [16] Briefly, individual semen samples were allowed to liquefy at room temperature of 25°C, and the sperm was separated from seminal plasma, immature germ cells, and non-sperm cells through density gradient centrifugation (300g for 20 min) using PureSperm® solution (Nidacon, Gothenburg, Sweden). The sperm pellet containing normal sperm was washed twice using phosphate buffered saline (PBS), then capacitated sperm was prepared by incubation of the previously washed sperm in Ham's F10 medium (Sigma, Germany) supplemented with 3.5% human serum albumin for 3 h at 37°C, and the aliquot was used freshly in subsequent techniques. ...
Article
Full-text available
Objective: Fibronectin (FN) is a multifunctional diametric glycoprotein on the surface of the sperm that plays an important role in the sperm-oocyte interaction and fertilization process. The aim of the present study was to assess the FN levels as a sperm surface biomarker for sperm selection in assisted reproductive technology. Material and methods: Polyclonal antibody against human FN was produced in rabbit. Its quality, purity, and immune reactivity were assessed by SDS-PAGE and western blot. In addition, the presence of FN on the sperm surface was assessed through immunocytochemistry and flow cytometry. The amount of FN and the sperm quality were assessed in normozoospermia (N) (42 men) and asthenoteratozoospermia (AT) (72 men) groups through sperm chromatin dispersion (SCD), sperm chromatin structure assay (SCSA), and chromatin maturation index (CMI). Results: The results showed the distribution of FN protein on the equatorial region of the human sperm surface. In addition, the FN levels were found to have a significant difference between the two groups with 24.64±9.08% in N and 16.90±7.27% in AT (p≤0.0001). In addition, FN level negatively correlated with SCD (p≤0.0001), SCSA (p≤0.0001), and CMI (p≤0.001). Threshold values of FN level and DNA fragmentation index (DFI) percentage were 16 and 30 and were identified as cut-off values to determine the N group with a specificity of 83.3% and 81.0% and a sensitivity of 16.8% and 19.0%, respectively. The specificity and sensitivity of FN-DFI were 91.2% and 8.8%, respectively. Conclusion: It appears that FN can be used for the selection of sperm with suitable quality, although future studies are recommended.
... For the analysis of sperm motility, the computer-assisted semen analysis system was used. [16] Briefly, individual semen samples were allowed to liquefy at room temperature of 25°C, and the sperm was separated from seminal plasma, immature germ cells, and non-sperm cells through density gradient centrifugation (300g for 20 min) using PureSperm® solution (Nidacon, Gothenburg, Sweden). The sperm pellet containing normal sperm was washed twice using phosphate buffered saline (PBS), then capacitated sperm was prepared by incubation of the previously washed sperm in Ham's F10 medium (Sigma, Germany) supplemented with 3.5% human serum albumin for 3 h at 37°C, and the aliquot was used freshly in subsequent techniques. ...
Article
Full-text available
STUDY QUESTION: Does Fibronectin (FN) protein as a new biomarker can be used for human sperm selection in ART? PARTICIPANTS/MATERIALS, SETTING, METHODS: Polyclonal antibody against human FN was produced in rabbit. Its quality, purity and immune reactivity were assessed by SDS-PAGE and western blotting (WB). Also presence of FN on sperm surface was assessed through immunocytochemistry (ICC) and flow cytometry (FCM). SUMMARY ANSWER: FN can be used for selection of sperm with suitable quality. WHAT IS KNOWN ALREADY: Specific antibodies, as powerful ligands for detection, separation and measurement of other suggested biomarkers were used in different studies. FN is a multifunctional diametric glycoprotein on the surface of sperm plays an important role in sperm-oocyte interaction and fertilization process. STUDY DESIGN, SIZE, DURATION: Amount of FN and the sperm quality were assessed in normozoospermia (N) (42 men) and asthenoteratozoospermia (AT) (72 men) groups through Sperm chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and chromatin maturation index (CMI). Semen samples were collected after 48–72 h of sexual abstinence and assessed according to World Health Organization guideline (WHO). MAIN RESULTS AND THE ROLE OF CHANCE: The results showed the FN distribution on the equatorial region of human sperm. Statistically significant differences were found in the FN levels of sperm surface between two groups with 24.64±9.08% in N and 16.90±7.27% in AT (p≤0.0001). Also, FN level correlated negatively with SCD (p≤0.0001), SCSA (p≤0.0001), and CMI (p≤0.001). A threshold of FN level and DFI percentage respectively were 16 and 30, were identified as a cut-off value to determine N with specificity 83.3% and 81.0%, and sensitivity of 16.8% and 19.0%. A specificity and sensitivity of FN-DFI were 91.2% and 8.8%. LIMITATIONS, REASONS FOR CAUTION: This study implied that the FN levels in sperm could be potentially used as a biomarker in sperm selection and assessing the quality of sperm in ART. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that FN can be used for selection of sperm with suitable quality although future studies are recommended.
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A method combining Janus green B and Thymol blue stains the anterior part of the head, the nuclear membrane, middle piece, and tail of spermatozoa light green and the nucleus deep purple. The method provides excellent stained preparations for the evaluation of sperm morphology by phase contrast microscopy. It produces significantly less abnormal spermatozoa compared with the Papanicolaou stain.
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This study on the epidemiology of human fertility emphasizes the study and analysis of several parameters. These include: the measure and distribution of fertility. The incidence of sterility is low (3-5% of couples) and the fecundability of fertile couples is approximately 30% per cycle. Approximately 7% of newly-formed couples per year will undergo complex treatment for infertility. The results of clinical and diagnostic explorations. Among infertile couples, the woman is responsible in approximately 60% of cases, and the man in approximately 25% of cases, and both of these factors may be associated. Clinical and diagnostic explorations are negative in approximately 18% of couples and the infertility is termed idiopathic. 'Normal' sperm characteristics vary according to age, seasonal or environmental factors. Female factors varying as a function of age, menstrual cycle, ovulation and functional status of the genital organs. Infertility in both partners leads to specific difficulties for epidemiological analyses, where the base unit is not an individual.
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Change in the nuclear maturity has been considered as an index of sperm quality. This is done by using aniline blue staining that stains spermatozoa with disturbances in the chromatin condensation. Therefore, this technique was used to evaluate the quality of sperm obtained by sperm preparation methods. In 14 semen samples from healthy donors with normal semen analysis and normal forms, the swim-up (SU), Percoll gradient centrifugation (PG), glass wool column filtration (GW) and sedimentation-migration (SM) were performed. GW and SM methods presented the lowest number of spermatozoa with alteration in the nuclear maturity (ANM), 11, 14% and 12, 42% compared to native semen (19%) (P < 0.005 and P < 0.05 respectively). SU and PG did presented no significant difference compared to native semen. In tests using the four methods, approximately 60% of the ANM occurred in normal spermatozoa. In the cells with pathologic abnormalities and ANM, 74.5% corresponded to macrocephalic forms, as this abnormality correlated mainly with ANM. It is concluded that in a semen sample with a higher percentage of normal forms, approximately 19% will have an ANM. GW and SM methods obtain the lowest percentage of ANM. ANM presents itself in 98% of the macrocephalic forms and they are effectively isolated with the PG method. The practice of this simple technique may orientate towards the sperm preparation methods to be used in assisted reproduction.
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In order to improve the selection of couples for intrauterine insemination (IUI) because of longstanding primary infertility of alleged male origin, we have performed a prospective study measuring conventional and advanced analysis of sperm characteristics, the hypoosmotic swelling test, the Shorr stain, the acidified aniline blue stain and alpha-glucosidase activity in seminal plasma, of 89 couples with no demonstrable abnormality of the female partner. Twenty-four couples attained spontaneous conception, 23 were successful within six cycles of IUI, and 42 remained without conception in spite of IUI during six unstimulated cycles. The proportion and concentration of spermatozoa with progressive motility was significantly lower (P < 0.01) in the successful IUI cases than in the couples attaining spontaneous conception, and the lower quartile value was lower in the former than in the latter. There were less pregnancies among IUI treated couples when sperm concentration and motility were within the range of normal fertile men, or when the concentration of white blood cells was elevated. More pregnancies occurred when markers of epididymal function, namely the result of the Shorr stain and alpha-glucosidase measurement, were normal. Total progressive motility and the result of the Shorr stain were the only independent variables selected by logistic regression to discriminate between successful and failed IUI cases. It is concluded that only a limited group of couples may benefit from IUI.
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The purpose of this study was to investigate the effect of different staining and washing procedures on the results of human sperm morphology evaluation by manual and computerised methods. Furthermore, it was intended to find the staining and washing combination which would provide optimal readability for computer-assisted sperm morphology evaluations. In phase one, four staining methods were evaluated for smears prepared from the resulting samples following a two times washing procedure. In phase two, 20 semen samples were used to compare the Diff-Quik and Papanicolaou staining methods, following one and two washes. All manual readings, of Papanicolaou and Diff-Quik stained smears, were comparable with each other, with means between 7.3% and 7.9% normal spermatozoa. All the manual readings were also comparable to the computer readings of the Diff-Quik slides following one and two washes with means of 9.0% and 5.9%, respectively. However, due to the higher computer readings found for the Papanicolaou stained smears, with means of 13.9% and 13.5% following one and two washes, respectively, a statistically significantly difference between overall computer and manual readings was found (Wilks' Lamda, P = 0.0002). Taking all data into consideration, it could be concluded that the one wash Diff-Quik stained smears was the optimal preparation method for computerised sperm morphology evaluation, comparing favourably with manual evaluations.
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To evaluate the importance of interindividual variations in the disomy frequencies of human sperm and their possible correlation with the principal semen parameters. Prospective randomized analysis of sperm nuclei by fluorescence in situ hybridization and analysis of semen parameters. University-based laboratory for reproductive biology. Fifty-seven human ejaculates selected at random from a population of men undergoing semen analysis. Semen specimens were analyzed, and sperm samples were prepared for fluorescence in situ hybridization. Semen parameters, including necrozoospermia, global motility, sperm concentration, multiple abnormalities index, and teratozoospermia were evaluated, aniline blue staining was completed, and disomy frequencies for chromosomes 8, 15, 18, X, and Y were determined using fluorescence in situ hybridization. Noticeable differences in disomy frequencies between individuals were observed, and these frequencies were correlated with the degree of nuclear maturity. We hypothesize that the positive correlation can be explained by an abnormality of chromosomal segregation at the time of meiosis that would cause disturbances during the transition of nucleoprotein or by one or several premeiotic abnormalities of chromatin that would perturb both the meiotic process and the construction of definitive proteins.
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Percentage and types of morphological abnormalities found in canine spermatozoa were evaluated by three investigators using three stains (Giemsa-Wright stain [Diff-Quik], eosin Y/nigrosin [Hancock], and eosin B/nigrosin [Society for Theriogenology morphology stain] with conventional light microscopy, compared to phase contrast microscopy on unstained samples. The percentage of spermatozoa with abnormal heads, midpieces, and tails varied by technique and by investigator. Average percentages of morphologically normal spermatozoa were significantly higher in samples stained with Diff-Quik and samples examined by phase contrast microscopy than in samples stained with Hancock or Society for Theriogenology morphology stains. No effect of investigator on the percentage of morphologically normal spermatozoa was assessed. Results suggest that staining or preparation technique may alter the morphology of canine spermatozoa artifactually.
Article
Automated sperm morphology analysis (ASMA) technology has improved the assessment of sperm morphology, but the results depend on the use of adequate and standardized procedures. In this study the Sperm-Class Analyzer (SCA) ASMA system was used to assess sperm head morphometry in the Cynomolgus monkey and to evaluate the influence of sample size, intraslide variation, and the use of three staining techniques on the accuracy of image processing and sperm head morphometry. Haematoxylin is the staining technique of choice for Cynomolgus spermatozoa, as optimum contrast of sperm heads with the surrounding background allows efficient segmentation, i.e. sperm head boundary detection, making the image analysis process more accurate. The analysis of 100 spermatozoa is recommended since a larger sample size did not result in more accurate sperm head morphometry. There were no differences in either the percentage of correctly binarized sperm heads or sperm head dimensions among samples obtained from different zones of the slides, although differences in stain intensity (grey level) were detected. The measurements made on Haematoxylin, Diff-Quik and Hemacolor-stained slides yielded different values for all of the sperm head parameters under consideration. This result demonstrates that the procedures of fixation and staining significantly affect the dimensions of sperm heads.
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A case control study was carried out to determine the value of sperm chromatin condensation in the assessment of male fertility. A total of 165 semen samples from 90 patients (cases) and 75 healthy donors (control) were examined for chromatin condensation (aniline blue staining), as well as conventional sperm parameters, notably sperm morphology, sperm count, and progressive motility. Whereas only 55 +/- 12.0% of the samples from the infertile patients were unstained by aniline blue (chromatin condensed), 78 +/- 19.0% of the samples in the control group did not take up the stain (chromatin condensed). A significant difference (p < .001) was observed between the two groups. Similarly, the difference between the mean percentage of morphologically normal spermatozoa for the infertile patients (12.1 +/- 1.2%) and the control (23.9 +/- 1.9%) was very significant (p < .001). In addition, only 50 out of the 90 patients (55.5%) had a normal sperm count, whereas all the 75 (100%) were normal in the control group. By comparing between the two groups a significant difference (p < .001) was also observed. Furthermore, a significant difference (p < .001) was also found between the cases and the control with regard to the percentage of spermatozoa illustrating linear progressive motility (40 +/- 9.7% vs. 70 +/- 12.3%). However, no correlation was found between sperm chromatin condensation and morphology, count, and motility. This study suggests that chromatin condensation constitutes a valuable parameter in the assessment of male fertility, completely independent of conventional sperm parameters. Consequently, the inclusion of chromatin condensation to routine laboratory investigations of semen prior to assisted reproduction is strongly recommended.