Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
Received: 2016.06.16
Accepted: 2016.08.10
Published: 2017.04.16
2785 — 5 28
Sleep Deprivation and Late Bedtime Impair
Sperm Health Through Increasing Antisperm
Antibody Production: A Prospective Study of
981 Healthy Men
ACDE 1 Mei-Mei Liu
BCF 2 Li Liu
BE 1 Liang Chen
C 3 Xiao-Jing Yin
B 1 Hui Liu
F 1 Yan-Hua Zhang
D 1 Pei-Ling Li
B 4 Shan Wang
D 4 Xiao-Xiao Li
D 4 Cai-Hong Yu
Corresponding Author: Mei-Mei Liu, e-mail: mm7723@yeah.net
Source of support: This study was supported by the Harbin Scientific Innovation Project for Elite Young Researchers (Grant No. 2013RFQXJ096)
Background: The aim of this study was to investigate the effects of sleep duration and bedtime on sperm health, and the
possible mechanism involved.
Material/Methods: We randomly divided 981 healthy Chinese men into groups according to research-set bedtimes (A=8–10 PM,
B=after 10 PM, and C=after midnight) and sleep durations: group 1=<6.0 h (short), group 2=7.0–8.0 h (aver-
age), and group 3=>9.0 h (long). Sperm morphology, count, survival, and motility were examined according to
sleep patterns. Antisperm antibody (ASA) production in semen was determined.
Results: Sperm counts and their survival rates were lower in the short sleepers as compared to others within each group
(all P<0.01). The lower counts and survival rates were observed in different bedtimes, with significant differ-
ences found between measurements of C1 vs. A1 and C2 vs. A2 or B2 (all P<0.05 or 0.01). Semen motility was
lower in the short sleepers as compared to the average and long sleepers (all P<0.01). There were differenc-
es in the bedtime-related results between measurements of C1 vs. A1 or B1 (P<0.05 or 0.01). Additionally, the
population proportion for the ASA-positive participates and incidence of the ASA-expressed population obvi-
ously increased in the short sleepers as compared to others within each group (all P<0.05).
Conclusions: Short and long sleep durations and late bedtime were associated with impaired sperm health in the study co-
hort, partly through increasing ASA production in the semen.
MeSH Keywords: Semen Analysis • Sperm Count • Survival Rate
Full-text PDF: http://www.medscimonit.com/abstract/index/idArt/900101
Authors’ Contribution:
Study Design A
Data Collection B
Statistical Analysis C
Data Interpretation D
Manuscript Preparation E
Literature Search F
Funds Collection G
1 Department of Obstetrics and Gynecology, Second Affiliated Hospital Harbin,
Harbin Medical University, Harbin, Heilongjiang, P.R. China
2 Department of Obstetrics and Gynecology, Harbin Red Cross Central Hospital,
Harbin, Heilongjiang, P.R. China
3 Department of Obstetrics and Gynecology, Chaoyang Central Hospital, Chaoyang,
Heilongjiang, P.R. China
4 Department of Obstetrics and Gynecology, Harbin Medical University, Harbin,
Heilongjiang, P.R. China
e-ISSN 1643-3750
© Med Sci Monit, 2017; 23: 1842-1848
DOI: 10.12659/MSM.900101
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Background
Sleep is a natural and periodic state of rest for the mind and
body, occurring at regular intervals. Proper sleep is a funda-
mental component of a healthy lifestyle. The amount of sleep
each person needs depends on many factors, including age.
For most adults, 7 to 8 h per night appears to be the best
amount of sleep [1–3]. Sleep deprivation is now being recog-
nized as an increasingly common condition inherent to mod-
ern society [4], so sleep loss not only is a social issue but also
leads to a large number of human disorders, including male
reproductive impairment [5]. It has been found that the fre-
quency of sleep disturbances has increased in the industrial-
ized world during the past few decades, and inadequate sleep
can decrease semen quality [6,7].
Some studies have shown that sleep deprivation may impair
immune function; the levels of IgG, IgA, and IgM were enhanced
in the sleep-deprived cohort, suggesting that insufficient sleep
may induce changes in the parameters of serum humoral im-
munity [8,9]. Despite the growing literature on the relation-
ship between sleep loss and reduced sperm quality [5,7], the
effects of sleep duration and bedtime on ASA production have
been studied less extensively.
The purpose of this study was to investigate effects of sleep
duration and the research-set bedtimes on affecting sperm
quality and the ASA production in 981 healthy Chinese men
living in the northeast region of China. Our results not only in-
dicated the effects of inappropriate sleep experiences on in-
fluencing sperm health, but also revealed an important mo-
lecular mechanism involved.
Material and Methods
Study population and inclusion criteria
In this prospective study, 981 healthy male adults voluntari-
ly participated in an investigation on the effects of sleep du-
ration and bedtime on i sperm quality during the period from
September 2013 to April 2014. The volunteers, ages 18–50
years (mean 29.56+5.38 years), were selected for this study
according to the following criteria: 1) a good and regular sleep
duration and ideal bedtime established over the last 6 months
prior to starting the study; 2) no clinical manifestations of neu-
rological or psychiatric disorders, and no history of trauma or
surgical treatments; 3) no medical conditions in which a man
does not have any measurable level of sperm in his semen
or there is no sperm in the ejaculate; 4) no medical problems
such as chronic systemic disease, endocrine disease, urinary
tract infections, or varicocele; 5) no prescribed drugs taken
within 1 month prior to the study; and 6) written informed
consent for using the sperm. This study was approved by the
Ethics Committee of the Second Affiliated Hospital of Harbin
Medical University.
Design of experiments
In this prospective study, we randomly divided 981 healthy
male adults into groups according the research-set bedtimes
and the length of sleeping time (in hours). All participants had
been in consistent sleep patterns for 3 consecutive months.
The volunteers in groups A, B, and C started to sleep at bed-
times of 8–10 PM, 10 PM–midnight, and after midnight, re-
spectively. The participants in each group were subdivided
into 3 subgroups based on duration of sleep (in hours): <6.0
h (short sleepers), 7.0–8.0 h (average sleepers), and >9.0 h
(long sleepers). The numbers of participants in each group
were 110, 105, and 113 in A1–3; 106, 111 and 114 in B1–3;
and 111, 104, and 107 in C1–3.
Preparation of semen samples
Semen collection refers to the process of obtaining semen
from male humans via masturbation, which is the most com-
mon way to collect a semen sample [10]. All donors were re-
quired to refrain from any sexual activity for 3–7 days before
collecting semen at our Hospital Reproduction Health Center,
since longer or shorter periods of abstinence may result in a
lower sperm count or decreased sperm motility. After wash-
ing hands with soap and water, all donors collected a semen
specimen by masturbation into a wide-mouth sterile sample
cup in a room close to the semen laboratory. The donors were
instructed to capture the first part of the ejaculate in the pro-
cess of collecting a semen sample and to not attempt to col-
lect any spilled semen. They were told to cap the container
as soon as a sample collection has been finished. The sam-
ple cups were rapidly placed in a water tank at 37°C. Semen
tests were not carried out until samples fully turned to liquid.
Semen analyses
A semen analysis typically measures the number of sperm per
milliliter of ejaculate, and analyzes sperm morphology and mo-
tility. In order to achieve the most accurate results, the semen
specimens obtained by masturbation were delivered to the lab
for semen analysis within 30 min of the specimen collection
time. The semen analysis was performed by use of a comput-
er image analyzer system (Cellsoft; Cryo Resources Co., NY).
The outcomes of analysis mainly included: 1) Sperm morphol-
ogy, which was viewed under a microscope (Olympus, Japan)
at a magnification of 400 and was expressed as a percent-
age of normal sperm having an oval head with a long tail; 2)
Sperm count, expressed as the number of sperm per milliliter
(mL) of semen in 1 ejaculation; 3) Survival rate, calculated as
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a percentage of the sperm with movement characteristics in
total sperm counts in the semen; and 4) Sperm motility, which
is sperm swimming forward progressively, expressed as a per-
centage of motile sperm according to WHO criteria [11]. More
specified measures were defined by motility grades of A (pro-
gressive motility) and B (non-progressive motility).
Assay of antisperm antibody
The ASA test was used to identify a special protein (antibody)
that attacks sperm. The kit for ASA detection was purchased
from Yilikang Biochemistry Technology Co., LTD (ZheJiang,
China). Content of the ASA production in the semen was mea-
sured by enzyme-linked immunosorbent assay (ELISA) accord-
ing to the manufacturer’s instructions. Briefly, 100 ml of semen
sample was added into the ELISA plate coated with a primary
antibody (1: 100) for human spermatozoa protein and then in-
cubated for 30 min at 37°C. After washing, horseradish perox-
idase-conjugated secondary antibody (1: 200) was added into
each well of the plate and incubated for 30 min. After washing,
50 ml of a substrate TMB (3,3’,5,5’-tetramethylbenzidine) was
added to yield a color reaction. Absorbance values were de-
termined at a wavelength of 450 nm using a microplate read
-
er. The experimental operation and analysis of results were
implemented strictly in accordance with the kit instructions.
Statistical analyses
Values are expressed as Mean ± Standard Deviation (SD).
Results were calculated as absolute numbers and a percent-
age of sperm counts in semen samples harvested from the
grouped participants. Statistical analysis was performed us-
ing the SAS 9.1 software package. Comparisons from groups
with individual measurements were performed by Fisher’s least
significant difference (LSD) or Dunnett’s test. The chi-square test
(c2) was conducted to analyze the significance of parameters
within groups. A P value of <0.05 was considered significant.
Results
Age distribution and sperm morphology
In this prospective study, 981 healthy male adults were select-
ed for assessing the effects of sleep conditions on sperm pro-
duction. Age distribution (in years) was first examined in the
grouped participants and the results are shown in Figure 1A.
In contrast, a similar distribution model for the average age
may clearly be observed at the ranges of 28.93–30.16 years in
the investigated participants. There were no statistically sig-
nificant differences found in the values of average ages be-
tween any 2 groups.
Sperm morphology in sleep conditions was examined under
a microscope. The numbers of normally shaped sperm in se-
men samples from the grouped participants was calculated
as a percentage (%) in a total sperm count and the results
are shown in Figure 1B. An average value (%) for the normal
sperm was displayed at the ranges of 68.27–70.71 years in
all groups. In further observation, the sperm cells in the se-
men were abundant in reaching over 68% of the total sperm
count in the study cohort. In contrast, there were no signifi-
cant differences in sperm morphology between any 2 groups.
Sperm count and survival rate
Sperm counts in the semen obtained from the grouped partic-
ipants were examined according to sleep patterns. Sperm cells
50
40
30
20
10
0
Age ( year)
AB C
123
100
80
60
40
20
0
Normally shaped sperm (%)
AB C
A B
Figure 1. Age distribution and sperm morphology. Age distribution for the grouped participants was examined in sleep conditions with
an average age (A) presented for the study cohort. Changes in sperm morphology were calculated as a percentage (B) of
normally shaped sperm cells in the total sperm counts. Data are expressed as Mean ±SD. A, B, and C were the research-set
bedtimes. Sleep duration is grouped as 1, 2, and 3.
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(million/ml) in the semen are expressed as an absolute count
in the semen volume and the results are shown in Figure 2A.
In data analysis, there were significant decreases in the val-
ues of A1, A3, B1, B3, and C1 as compared to A2, B2, and C2
within each group (all P<0.01). Furthermore, the sperm counts
in groups C1 and C2 significantly decreased with between C1
vs. A1 and C2 vs. A2 or B2 (all P<0.01).
Survival rates for sperm cells in the semen were examined with
sleep experiences in the grouped participants and the results
are shown in Figure 2B. In statistical analysis of the survival
rate, there were obvious decreases in the values from the A1-,
A3-, B1-, B3- and C1-grouped cohorts as compared to others
within each group (all P<0.01). Moreover, a significant decrease
in the survival rate was also observed in the C2-grouped partic-
ipants with a difference between C2 vs. A2 or B2 (both P<0.05).
Observation on sperm motility
Sperm motility at levels A and B was analyzed according sleep
conditions. Data regarding the motility were calculated as a
percentage in the total sperm cells in each group and the re-
sults are shown in Figure 3A and 3B. In analysis of the A lev-
el, there were significant lower values of A1, A3, B1, B3, and
C1 as compared to others within each group (all P<0.01).
Additionally, there were significantly lower sperm counts in
the C1-grouped participants, with significant differences be-
tween C1 vs. A1 or B1 (both P<0.05). In terms of the B level,
150
125
100
75
50
25
0
Sperm count (million/ml)
A
**
$$
##
**
**
BC
100
80
60
40
20
0
Survival rate sperm (%)
A
** **
#
**
**
BC
123
A B
Figure 2. Sperm count and survival rate. Sperm counts (A) in semen samples and their survival rates (B) were examined in sleep
patterns in the grouped participants. Sperm counts (million/ml) and survival rates (%) are shown as an absolute number of
sperm cells in the semen volume. The results are expressed as Mean ±SD. A, B, and C were the research-set bedtimes. Sleep
duration is grouped as 1, 2, and 3. ** P<0.01 vs. A1, A3, B1, B3, and C1. #, ## P<0.05 vs. A2 and B2 in survival rate or <0.01 vs.
A2 and B2 in sperm counts. $$ P<0.01 vs. A1.
30
25
20
15
10
5
0
Population (A) of motile sperm (%)
A
** **
**
$
**
BC AB C
50
40
30
20
10
0
Population (B) of motile sperm (%)
** **
$$
** ##
**
123
A B
Figure 3. Sperm motility. Sperm motility at the levels of A and B was tested in sleep conditions. Data were calculated as a percentage
in the total sperm cells in each group of participants. The results are expressed as Mean ±SD. A, B, and C were the research-
set bedtimes. Sleep duration is grouped as 1, 2, and 3. ** P<0.01 vs. A1, A3, B1, B3, and C1 in the A level and vs. A1 and C1 in
the B level. ## P<0.01 vs. A2. $, $$ P<0.05 or 0.01 vs. A1 and B1 in the A level and vs. B1 in the B level.
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there were significantly lower values of A1 and C1 as com-
pared to others within each group (all P<0.01). In further anal-
ysis, obviously lower levels were observed in the C1- and C2-
grouped cohorts, with significant differences between C1 vs.
B1 and C2 vs. A2 (P<0.05 or 0.01).
Demographic distribution and incidence of ASA-positive
participants
Demographic data from the grouped volunteers were collected
from groups with different sleep conditions. The numbers of
the grouped participants were shown at the ranges of 104–114
(Figure 4). Population proportions of ASA-positive participants
were significantly higher in the A1-, B1-, and C1-grouped co-
horts as compared to others within each group (all P<0.05).
In comparison of the proportions from groups A1, B1, and C1,
there were no significant differences in the proportional dis-
tributions of ASA-positive individuals between any 2 groups.
The proportion of ASA-expressing participants was calcu-
lated as a percentage in the total population in each group
(Figure 5). In contrast, the numbers of ASA-positive individu-
als in groups A1, B1, and C1 were significantly higher (2-fold)
within each group (all P<0.05). In further analysis of A1, B1,
and C1, there were no significant differences in the incidence
between any 2 groups.
Discussion
Sleep is essential for mental and physical health. Human sleep
needs vary by age and among individuals; therefore, the ef-
fect of age must be accounted for in research on the effect
of length of sleep on male fertility [12,13]. Demographic data
obtained from these healthy male adults manifested a similar
distribution pattern of the average age with a slight change
of about 2 years among the grouped participants. In con-
trast, there was no significant difference seen between any 2
groups, indicating that the age factor did not need to be con-
sidered in further measures and statistical analysis regarding
these effects of sleep experiences on sperm health in the in-
vestigated cohort.
Sperm morphology is used to observe the size and shape of
sperm cells [14], which is one of the semen examinations in
evaluating the effects of sleep on sperm production. Our re-
sults showed that the percentages of normally shaped sperm
in the total sperm count reached 68.27–70.37%. Furthermore,
there was no significant difference observed between any 2
groups, indicating that differences in sperm morphology are
not a sensitive parameter for assessing the effects of sleep
conditions on sperm quality.
Figure 4. Distribution for ASA-positive
population. Demographic data from
the grouped participants was analyzed
in sleep conditions in presence
(black) and absence (white) of ASA
production. A population proportion
for the ASA-positive participants
presented as individual numbers in
each group. A, B, and C indicate the
research-set bedtimes. Sleep duration
is grouped as 1, 2, and 3. A c2 test
showed a p value of <0.05 vs. others
within each group, respectively.
* P<0.05 vs. other incidences within
each group.
A1
ASA+
ASA–
A2 A3
B1 B2 B3
C1 C2 C3
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
ASA+
ASA–
Figure 5. Incidence of ASA-positive individuals. Proportion of
ASA-positive individuals was examined according to
sleep conditions in the grouped participants, expressed
as a percentage of the total participants in each
group. The results are shown as Mean ±SD. A, B, and
C indicate the research-set bedtimes. Sleep duration
is grouped as 1, 2, and 3. * P<0.05 vs. other incidences
within each group.
Incidences of cohorts with ASA+ (%)
***
1
AB C
23
30
25
20
15
10
5
0
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Sperm count and survival rates were analyzed according to
sleep patterns. Our data revealed that the numbers of sperm
cells in the semen and their survival rates were significant-
ly lower in all of the short sleepers and the most of the long
sleepers, suggesting that reduction in the sperm cells depend
-
ed on the length of sleep time. It has been reported that short
and long sleep were associated with a higher prevalence of
coronary heart disease, stroke, and diabetes, in addition to
obesity and frequent mental distress, compared with opti
-
mal sleepers [15,16]. Lack of sleeping time may reduce sperm
quality [7]. However, an extra hour of sleep in this study also
showed lower sperm count and survival rate, suggesting the
negative effect of sleeping too little or too much. Furthermore,
sleeping longer does not necessarily produce higher-quality
sleep. For most adults, the optimal length of sleeping time is
about 7–8 h a night [1–3]. Long sleeping commonly is >9.0 h
of sleep each night for adults [17], which may disrupt circadi-
an rhythms, affecting the depth and length of sleep on sub-
sequent nights, thereby impairing sperm health.
In contrast to the individuals who went to bed at 10 PM-
midnight, our data clearly show that late bedtime is associ-
ated with lower sperm counts and survival in all of the short
sleepers and some of the average sleepers, suggesting that
late bedtime is an important factor. Frequent shifts in sleep
timing were associated with metabolic problems and increased
prevalence of depressive symptoms [18,19]. However, the lit-
erature on effects of bedtime on sperm health is scant. Based
on the effect of late bedtime on reduction of sperm produc-
tion, it is likely that a later bedtime could reduce the depth of
sleep, which in turn can lead to poor sleep quality on subse-
quent nights due to feeling sleepy the next day.
In support of the above findings, sperm motility at the A and
B levels were examined in sleep conditions. The data from
the A level showed that the proportion of progressively mo-
tile sperm was significantly lower in all of the short sleepers
and most of the long sleepers, which was identical to the re-
sults on the sperm count and survival. In the B level, our re-
sults show that the numbers of non-progressively motile sperm
were lower only in most of the short sleepers, suggesting that
impairment of sleep had less effect on sperm motility at the
B level than on the A level. In association with these findings,
our results also revealed that a late bedtime decreased the
numbers of motile sperm at both levels, suggesting that later
bedtime reduced sperm movement. Numerous studies have
shown that sperm motility can be affected by unhealthy life-
style [20,21]. Moreover, a poor sleep schedule can make affect
the quantity and quality of sleep [22,23]. The lower sperm mo-
tility is probably due to a ripple effect of the bedtime-induced
poor sleep quality.
A possible mechanism by which sleep experiences influenced
sperm quality was investigated in the grouped participants.
Our results showed a similar distribution pattern of popula-
tion proportions for the average and long sleepers who were
ASA-positive. There was a significantly higher proportion of
ASA-positive individuals among short sleepers, with no dif
-
ferences in bedtimes between the grouped cohorts, suggest-
ing that the increased ASA production was associated with
shorter duration of sleep but not bedtimes. In association
with this finding, the proportion of individuals who were ASA-
positive was obviously higher in the short sleepers as com-
pared to others within each group, strongly supporting that
deficient sleep was a major contributor to presence of ASA in
the semen. Antisperm antibodies are immune-reactive parti-
cles produced by the body as a response against the proteins
contained in sperm [24]. The antibodies target sperm in the
body and destroy healthy sperm [25]. The male may produce
antibodies to his own sperm as a result of contact between
blood cells and sperm, as through testis inflammation, varico-
cele, intercourse, or sometimes for unknown reasons [26–28].
Our study revealed a relationship between insufficient sleep
duration and increased ASA content, and it would be useful
to know how sleep deprivation affects sperm health. Since the
lower ASA level in the short sleepers was not associated with
changes in the research-set bedtimes, it is likely that the effect
of bedtime in impairing sperm health was a separate factor,
with a potential effect on production of sperm cells. Further re-
search is needed to determine the exact mechanism involved.
Conclusions
Our study results show that short and long sleep durations
and later bedtime can reduce sperm count, survival, and mo-
tility, partly through increasing ASA production.
Acknowledgments
We would like to thank Dr. Zhuofu Liu for help with statisti-
cal analysis.
Conflict of interest disclosures
None.
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Liu M.-M. et al.:
Effect of sleep time on sperm quality
© Med Sci Monit, 2017; 23: 1842-1848
CLINICAL RESEARCH
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