Study question: What is the prevalence of
defects in the Ca2+-signalling pathways mediating
hyperactivation (calcium influx and store mobilization) among donors and
sub-fertile patients and are they functionally significant, i.e. related to
fertilization success at IVF?
Summary answer: This study identifies, for
the first time, the prevalence of Ca2+ store
defects in sperm from research donors, IVFand ICSI patients. It highlights
the biological role and importance of Ca2+ signalling
(Ca2+ store mobilization) for fertilization
at IVF.
What is known already: Sperm motility and
hyperactivation (HA) are important for fertility, mice with sperm incapable
of HA are sterile. Recently, there has been significant progress in our
knowledge of the factors controlling these events, in particular the
generation and regulation of calcium signals. Both pH-regulated membrane Ca2+ channels (CatSper) and Ca2+ stores (potentially activating store-operated
Ca2+ channels) have been implicated in
controlling HA.
Study design, size, and duration: This was a
prospective study examining a panel of 68 donors and 181 sub-fertile patients
attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and
ICSI. Twenty-five of the donors gave a second sample (_4 weeks later) to confirm
consistency/reliability of the recorded responses. Ca2+ signalling was manipulated using three
agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation
of CatSper channels, potentially enhancing mobilization of stored Ca2+ by CICR) and 4-aminopyridine (4-AP) (effect
on pH equivalent to NH4Cl and mobilizes stored Ca2+). The broad-spectrum phosphodiesterase
inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was
also used for comparison. For patient samples, an aliquot surplus to
requirements for IVF/ICSI treatment was examined, allowing direct comparison
of Ca2+ signalling and motility data with
functional competence of the sperm.
materials,
setting, methods: The donors and sub-fertile patients were screened for HA
(using CASA) and changes in intracellular Ca2+ were assessed by loading with Fura-2 and
measuring fluorescence using a plate reader (FluoStar).
Main
results and the role of chance: The relative efficacy of the stimuli in
inducing HA was 4-AP .. IBMX . progesterone.
NH4Cl increased [Ca2+]i similarly to 4-AP and progesterone but did
not induce a significant increase in HA. Failure of samples to generate HA
(no significant increase in response to stimulation with 4-AP) was seen in
just 2% of research donors but occurred in 10% of IVF patients (P = 0.025).
All donor samples generated a significant [Ca2+]i increase when stimulated with 4-AP but
3.3% of IVF and 28.6% of ICSI patients failed to respond. Amplitudes of HA
and [Ca2+]i responses to 4-AP were correlated
with fertilization rate at IVF (P = 0.029; P = 0.031, respectively).
Progesterone reliably induced [Ca2+]i
responses (97% of donors, 100% of IVF patients) but was significantly less
effective than 4-AP in inducing HA. Twenty seven per cent of ICSI patients
failed to generate a [Ca2+]i
response to progesterone (P = 0.035). Progesterone-induced [Ca2+]i responses were correlated with
fertilization rate at IVF (P = 0.037) but induction of HA was not. In donor
samples examined on more than one occasion consistent responses for
4-AP-induced [Ca2+]i (R2 = 0.97) and HA (R2 = 0.579) were
obtained. In summary, the data indicate that defects in Ca2+ signalling leading to poor HA do occur and
that ability to undergo Ca2+-induced
HA affects IVF fertilizing capacity. The data also confirm that release of
stored Ca2+ is the crucial component of Ca2+ signals leading to HA and that Ca2+ store defects may therefore underlie HA
failure.
Limitations,
reasons for caution: This is an in vitro study of sperm function. While the
repeatability of the [Ca2+]i and
HA responses in samples from the same donor were confirmed, data for patients
were from 1 assessment and thus the robustness of the failed responses in
patients’ needs to be established. The focus of this study was on using 4AP,
which mobilizes stored Ca2+ and is
a potent inducer of HA. The n values for other agonists, especially calcium
assessments, are smaller.
Wider implications of the findings: Previous
studies have shown a significant relationship between basal levels of HA,
calcium responses to progesterone and IVF fertilization rates. Here, we have
systematically investigated the ability/failure of human sperm to generate
Ca2+ signals and HA in response to targeted
pharmacological challenge and, related defects in these responses to IVF
success. [Ca2+]i signalling is fundamental for sperm
motility and data from this study will lead to assessment of the nature of
these defects using techniques such as single-cell imaging and patch
clamping.
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