Natural selection of
sperms in female genital tract out of normal coitus:-
How
many of you are aware that researchers have estimated that a median of 35% of
spermatozoa were lost through flow
back after natural coitus . Surprisingly same group of researchers have
noticed in healthy fertile volunteers that in 12% of copulations almost 100% of
the sperm inseminated were eliminated. That may be the reason why pregnancy do
not ensue in each month in healthy couple. This suggests that less than 1% of
sperm might be retained in the female reproductive tract and this supports the
notion that only a minority of sperm actually enters cervical mucus and Human males ejaculate a considerably large number of morphologically abnormal sperms .
Many studies have confirmed that of the millions of sperm inseminated at coitus
in humans, only a few thousand reach the Fallopian tubes and, ordinarily, only
a single sperm fertilizes an oocyte.
We now know that there are many
physiological barriers in the female genital tract which act as the natural selection
for best sperms . This knowledge is essential to unearth causes of repeated
preg losses, anembryonic preg and long standing subfertility. Passage
of sperm through the female reproductive tract is regulated to maximize the
chance of fertilization and ensure that sperm with normal morphology and
vigorous motility will be the ones to succeed.
Ejaculated sperms do contain millions of abnormal sperms which
include morphological abnormalities , motility disorders .These are principally due to abnormal DNA content of sperm .
The natural physiological barriers filter not all such abnormal DNA containg sperms as such there is
subfertility due to fertilization of oocyte with poor sperms..
Millions of sperms (both normal &
abnormal sperms) have to undergo many a obstacles in order to reach the oocyte
and finally fertilize the oocyte. These physiological
barriers initiating from the vaginal pH upto uterine fluid act as filters
allowing only best sperms to swim up .This happens normal fertile women. There is
the natural selection of the sperm and
any deficiency or abnormality at any level may allow a morphological defective
sperm to go inside the tube and fertilize. This often results in failure in fertilization,
blighted ovum, ectopic, missed abortion etc. As such nature has made a filter
which will allow morphologically best sperm to go in utero-fallopian fluid
fertilize the oocyte.
How many millions sperms are lost in
sperm migration process in the female genital tract?? Ans: Studies were on from early fifties and In 1992
it was definitely shown by different researchers that there was a
dramatic reduction in the number of sperms recovered from the fallopian tube
when artificially inseminated in volunteers.
This
reduction clearly brought about a change in the thought process at that
time. Researchers therefore are now focusing on details of the physiological barrier which
allows the natural selection that the sperm
. These physiological
barriers include the 1) acidic ph of
the vagina , 2) cervical mucus response, 3)
changes in the uterine
environment. At all these points
millions are sperms are filtered and only the best quality reaches the
F tube for fertilization at ampulla. Few millions are lost during passage
of the uterus to the tubes and finally
one sperm gets competency to bind the zona pellucida .
Sperm transport:- Theory A) Most of the sperms go right into cervical canal soon after
coitus. At coitus, human
sperm are deposited into the anterior vagina, where, to avoid vaginal acid and
immune responses, they quickly contact cervical mucus and enter the cervix.
Cervical mucus filters out sperm with poor morphology and motility and as such
only a minority of ejaculated sperm actually enters the cervix. Cervical mucus
presents a greater barrier to abnormal sperm that cannot swim properly or that
present a poor hydrodynamic profile than it does to morphologically normal,
vigorously motile sperm and is thus thought as one means of sperm selection .The
greatest barrier to sperm penetration of cervical mucus is at its border,
because here the mucus micro architecture is more compact .Components of
seminal plasma may assist sperm in penetrating the mucus border. More human
sperm were found to enter cervical mucus in
vitro when an inseminate was diluted 1:1 with whole seminal plasma
than when it was diluted with Tyrone’s medium, even though the sperm swam
faster in the medium In the uterus, muscular contractions may enhance passage
of sperm through the uterine cavity. A few thousand sperm swim through the
uterotubal junctions to reach the Fallopian tubes where sperm are stored in a
reservoir, or at least maintained in a fertile state and receive oxygen & nutrition
from tubal fluid.
As the time of
ovulation approaches, sperm become capacitated and hyper
activated, which enables them to proceed towards the tubal ampulla.
Sperm may be guided to the oocyte by a combination of thermotaxis and
chemotaxis.
Theory B:-The second theory of sperm transport is that
soon after ejaculation the semen get coagulated before sperms enter the cervical
canal. A large number of sperms are entrapped by
a coagulum, in the vagina where it coagulates to form a copulatory plug.
The plug forms a cervical cap that promotes sperm transport into the uterus Human
semen coagulates, but it forms a loose
gel rather than the compact fibrous plug. The coagulate forms within about
a minute of coitus and then is enzymatically degraded in liquid state by 20-30
minutes time . The predominant structural proteins of the gel are the 50 kPa semenogelin I and the 63 kPa semenogelin II, as well
as a glycosylated form of semenogelin II, all of which are secreted primarily
by the seminal vesicle.
Later
, the gel is degraded by
prostate-specific antigen (PSA), a serine protease secreted by the prostate
gland .It has been proposed that this coagulum serves to hold the sperm
at the cervical os
and that it protects sperm against the hostile
acid environment of the vagina Seminal
gels are not fully successful at holding sperm at the cervical os.
What is seminal effluents (‘flowback’).
?? The fate of spermatozoa that are
ejaculated or artificially inseminated into the vagina, but that do not enter
the cervix, has not been studied in details. But few researcher examined the volunteered who consented that characteristics
of sperm loss from the vagina following coitus (‘flowback’). They found that flowback occurred in 94% of copulations
with the median time to the emergence of ‘flowback’ of 30 min (range 5–120
min). Furthermore they estimated that a median of 35% of spermatozoa were
lost through flowback but that in 12% of copulations almost 100% of the sperm
inseminated were eliminated. This suggests that less than 1% of sperm might be
retained in the female reproductive tract and this supports the notion that
only a minority of sperm actually enters cervical mucus and ascend higher into
the female reproductive tract.
Human
sperm must contend, however briefly, with the acidic pH of vaginal fluid. The
vaginal pH of women is normally five or lower, which is microbicidal for many
sexually transmitted disease pathogens. Evidence indicates that the acidity is
maintained through lactic acid production by anaerobic lactobacilli that feed
on glycogen present in shed vaginal epithelial cells.
In
additions to pH buffers, seminal plasma
contains inhibitors of immune responses, including protective components that
coat sperm . These are most
effective when sperm are bathing in seminal plasma and may be gradually shed
when sperm leave the seminal plasma behind.
Males
may also overcome female defenses by inseminating many sperm. This strategy is
particularly effective for overcoming cellular immune responses.
Sperm of humans enter the cervical canal
rapidly where they encounter cervical mucus . Under the influence of estrogen
the cervix secretes highly hydrated mucus,. The extent of hydration is
correlated with penetrability to sperm . Coitus on the day of maximal mucus
hydration in women is more closely correlated with incidence of pregnancy than
coitus timed with respect to ovulation detected using basal body temperature
The role of
neutrophils in vaginal canal ?? .
It has been demonstrated that neutrophils present in vaginal fluid will bind to human sperm and ingest them only
if serum that contains both serological complement and complement-fixing
anti-sperm antibodies is present. This can happen in vivo if the female somehow
becomes immunized against sperm antigens. Altogether, the evidence indicates
that leukocytic invasion serves to
protect against microbes that accompany sperm and does not normally present a
barrier to normal motile sperm, at least not shortly after coitus.
What about role by Immunoglobulins??
Immunoglobulins, IgG and IgA, have been detected in human cervical mucus.
Secretory IgA is produced locally by plasma cells in subepithelial connective
tissue. The amount secreted increases in the follicular phase but then
decreases at about the time of ovulation. The vagina is open to the exterior
and thus to infection, especially at the time of coitus; therefore, it is well
equipped with antimicrobial defenses. These defenses include acidic pH and
immunological responses and can damage sperm as well as infectious organisms.
To enable fertilization to take place, both the female and the male have
adopted mechanisms for protecting sperm.
Complement
proteins are also present in cervical mucus along with regulators of complement
activity Thus, there is a potential for
antibody-mediated destruction of sperm in the cervical mucus as well as
leukocytic capture of sperm. Some anti-sperm antibodies are not
complement-activating; however, they can still interfere with movement of sperm
through cervical mucus by physical obstruction .
.
The immunoglobulins provide greater
protection from microbes at the time when the cervical mucus is highly hydrated
and offers the least resistance to penetration. However, when there are
antibodies present that recognize antigens on the surface of ejaculated sperm,
infertility can result (Immunological infertility)
Hyper activation is a special process adopted by good sperms and such assists sperm in penetrating
mucus in the tubes and the cumulus oophorus and zona pellucida of the oocyte,
so that such sperms may finally fuse with the oocyte plasma
membrane. Therefore knowledge of the biology
of sperm transport can inspire improvements in artificial insemination,
IVF, the diagnosis of infertility and the development of contraceptives.
What about
allogencity of sperm and how sperms overcome existing immune system in female
genital tract?? Ans:- we know sperm proteins are allogenic to
the female, they may encounter the defenses of the female immune system meant
for infectious organisms .Oocytes are usually fertilized within hours of
ovulation In humans, there is evidence that fertilization occurs when
intercourse takes place up to five days before ovulation (Wilcox et al.,
1995). Because sperm are terminally differentiated cells, deprived of an active
transcription and translation apparatus, they must survive in the female
without benefit of reparative mechanisms available to many other cells. Sperm
are subjected to physical stresses during ejaculation and contractions of the
female tract, and they may sustain oxidative damage. Furthermore, because sperm
are allogenic to the female, they may encounter the defenses of the female
immune system meant for infectious organisms .Thus, sperm must somehow use
their limited resources to maintain their fertility in the face of numerous
impediments.
Vaginal defenses against infectious
organisms may affect sperm
.
.
Furthermore, based on histochemical staining characteristics of the mucus, they
concluded that, during the follicular phase, mucus deep in the channels is
different in composition and less dense than that in the central portion of the
cervical canal. They proposed that bull sperm enter deep channels at the
external os and travel in them all the way to the uterine cavity, thereby
avoiding the more viscous mucus in the centre of the cervical canal that serves
to discharge uterine contents. This model is supported by results of earlier
studies on farm animals. . Normal, fresh, motile sperm can avoid the area most
populated by neutrophils and they appear to be resistant to leukocytic
phagocytosis anyway, as discussed above. In descriptions of human cervical
anatomy, mention is made of cervical crypts that are thought to entrap and
store sperm (scanning electron microscopy of the human cervix indicates that
mucosal grooves forming a preferential pathway for sperm could be present as in
the bovine (Figure 2).
A comprehensive study of the human cervix is needed to determine whether sperm
follow mucosal grooves to traverse the cervical canal.
Sperm
may also be guided through the cervix by the micro architecture of the cervical
mucus. Mucins, the chief glycoproteins comprising cervical mucus, are long,
flexible linear molecules (molecular weight of human mucins is approximately 107
Daltons). The viscosity of mucus is due to the large size of mucins, while
elasticity results from the entanglement of the molecules . It is thought that
these long molecules become aligned by the secretory flow in mucosal grooves
and thus serve to guide sperm. Human sperm have been demonstrated to orient
themselves along the long axis of threads of bovine cervical mucus. Human sperm
swimming through cervical mucus swim in a straighter path than they do in
seminal plasma
Are sperm stored in the cervix?
Little
is known about how long sperm spend traversing the cervix or whether sperm are
stored there. Vigorously motile sperm have been recovered from the human cervix
up to 5 days after insemination , and the presence of sperm in mid-cycle
cervical mucus forms the basis of the ‘post coital test’ (PCT) . Nevertheless,
it is not known whether sperm collected from cervices this long after coitus
would reach the Fallopian tube and succeed in fertilizing, nor could it be
known whether these sperm had re-entered the cervix from the uterus. Very few
sperm have been recovered from human uteri 24 h after coitus and those sperm are greatly outnumbered by
leukocytes
Unless
sperm are protected from phagocytosis (and they appear to be), it is unlikely
that they could travel from a cervical reservoir to the oviduct 24 h post
coitus.
.
Overall,
data of human sperm distribution in the Fallopian tubes of women have not
provided a clear picture of the events of sperm transport. Sperm recovered at
various times in different regions of the Fallopian tube have varied so much in
numbers that the data do not permit the construction of a model for the pattern
of tubal sperm transport . Nevertheless, since pregnancy has been shown to
result from intercourse as long as five days before ovulation (Wilcox et al.,
1995) human sperm must be stored somewhere in the female tract and the fact
that human endosalpingeal epithelium prolongs survival of sperm in vitro
indicates that the Fallopian tubes are strong candidates for storage sites.
In
addition to assisting sperm in reaching the oocyte, hyperactivation
also aids sperm in penetrating the zona pellucida. When hyperactivation
was blocked in capacitated, acrosome-reacted hamster sperm bound to the zona,
they were unable to penetrate it
There
is evidence for the existence of two complementary guidance mechanisms operating within the Fallopian
tube. The first (long-range) mechanism is where capacitated
sperm—released from intimate contact with the endosalpinx —are guided by thermotaxis
towards the site of fertilization. Once in the tubal ampulla, and at a closer
proximity to the oocyte, a second (short-range) chemotactic mechanism may guide
sperm closer to the oocyte . Sperm are equipped with a mechanism for turning
towards the oocyte in response to chemotactic factors; that is, they can switch
back and forth between symmetrical
flagellar beating and the asymmetrical flagellar beating of hyperactivation.
Hyper activation is reversible so sperm can alternate between turning and
swimming straight ahead. Mammalian sperm have been reported to Nevertheless,
the chemotactic agent in follicular fluid has not been identified, nor has its
presence in the Fallopian tube been detected. Odorant receptors unique to sperm
have been localized to a spot on the base of the flagellum of human .
Lessons for assisted conception
Assisted conception in humans and other
mammals relies on techniques to either assist sperm to reach the site of
fertilization (by techniques of insemination) or generate embryos in the
laboratory (by means of IVF) that can then be returned to the uterine cavity at
a later time. Although all of these techniques are well established, they do
not guarantee success. Understanding the biology of sperm transport and storage
in the female reproductive tract could inspire technological improvements in
assisted conception.
What about sperm DNA fragmentation ??
Researchers
have also noticed that failure to fertilize injected sperms in ICSI / Recurrent
early pregnancy loss may follow in women whose husband’s sperms exhibit have higher rates of DNA damage and a
higher occurrence of carrying
abnormal numbers of chromosomes .
Thus patients that have
a high DNA fragmentation index or partner have suffered
from recurrent miscarriages or
have poor quality of embryos
in multiple cycles can be benefitted from this
technique carried out a Cochrane review
in 2014 which came to the conclusion that more evidence is required to come to a conclusion to whether or not PICSI
is actually beneficial or not .
IMSI
How best to
avoid poor sperms (morphologically sperms) IMSI: Injection of
morphologically selected sperms: Humans ejaculate a considerably large number of
morphologically abnormal sperms . We have a strict criteria to grade the morphology of the sperm and also to decide on the treatment options for the patients. Most of the times
patients with poor morphology are
directed towards an ICSI cycle justification being that the embryologist
can choose the
morphologically normal sperms
to fertilise the oocyte . However recent papers have shown that even the sperms which appear normal in ICSI
can have multiple defects as shown
.
Gross evaluation
of sperms on ICSI
magnification that is 400x
isn’t sufficient to defect
subtle aberrations which according to some have higher chances of transmitting genetic as well as
chromosomal diseases . Also there is a need to standardise the scoring
system with IMSI as of now there are 4
grades in which the sperms can be
classified into the according to the current grading
more standardized . Cochrane review
on IMSI states that
there was no significant
difference in live birth between
IMSI and ICSI also
no significant difference in pregnancy rates or
miscarriages rtes But we need to
study the prognosis of an IMSI cycle vs
and ICSI cycle in patients who actually have poor parameters and then only
we would be able to justify the
use of IMSI.
as such
Sperm vacuoles
have also been researched and
have mostly been associated with a
higher DNA fragmentation index but
this association still needs to be proven
.
,Application of MACS in IVF
Improves fertilisation potential
Increases cleavage
and pregnancy rates
Reduces sperm DNA fragmentation
Improves live
birth rate
But again more systematic reviews are needed to back these
claims
MICROFLUIDICS
Microfluidics
is an exciting technology which
now takes into consideration the biophysical and biochemical milieu which
the sperm encounters in the female
reproductive tract. Studies
have shown an increase in motile
percentage of sperm from below
60% to 90% they have
also shown better DNA
integrity post sorting system
could reduce the treatment time for
intracytoplasmic .
Even though
the technological advances are occurring at a rapid pace the biggest challenge that still
remains is justifying these technologies and proving
their efficacy with
long term data to support it .
Sperm selection
brings forth a very exciting new frontier in IVF and it takes us closer to
achieving our aim of having
a single live birth from a single embryo
transfer. It also presents us with
various challenges as far as male infertility and its management is concerned.
Simple
insemination techniques, bypassing the vagina and cervix, have proved
advantageous for artificial insemination in dairy. Whereas, a bull normally
deposits several billion sperm into the vagina, artificial inseminators deposit
5–20 million frozen/thawed sperm directly into the body of the uterus leading to successful conception about 70%
of the time . Similar techniques are used in human females where washed
preparations of human sperm are inseminated directly into the uterine cavity or
the Fallopian tube . Often this is in combination with ovarian stimulation of
the female partner. Although moderate success rates can be achieved with these
techniques, it is unknown whether there are effects of sperm preparation or
ovarian stimulation on how sperm behave or respond to the biology of the female
reproductive tract before fertilization. Clearly, such information could be
useful for those who are interested in the improvement of these techniques.
Similarly,
there has been a long held assumption that, because fertilization can now be
achieved in
vitro, there must be little influence of the reproductive
epithelium on the physiology of gametes and embryos. However, in IVF many
thousand sperm are required to be incubated with oocytes in order to achieve
fertilization but evidence from most mammalian species suggest that only a few
sperm are present at the site and time of fertilization in the Fallopian tube This
staggering difference almost certainly reflects our inability to adequately
mimic in vitro
the environment and sperm selection mechanisms of the female reproductive tract
during natural unassisted conception. Again, clearly if we knew more of the
basic biology that underpins these events in
vivo then there is the potential for improvements to be made to IVF
procedures that could impact on the clinical outcome.
Finally,
diagnosis of the causes of infertility could be greatly improved if more were
known of the means by which sperm travel through the female reproductive tract
and the mechanisms that regulate the movement of sperm.
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