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STUDY QUESTION Is it possible to
restore ovarian function and natural fertility following the cryopreservation
and autotransplantation of whole ovaries, complete with vascular pedicle, in
adult females from a large monovulatory animal model species (i.e. sheep
SUMMARY ANSWER Full (100%)
restoration of acute ovarian function and high rates of natural fertility
(pregnancy rate 64%; live birth rate 29%), with multiple live births, were
obtained following whole ovary cryopreservation and autotransplantation
(WOCP&TP) of adult sheep ovaries utilizing optimized cryopreservation and
post-operative anti-coagulant regimes
WHAT IS KNOWN ALREADY Fertility
preservation by WOCP&TP requires successful cryopreservation of both the
ovary and its vascular supply. Previous work has indicated detrimental
effects of WOCP&TP on the ovarian follicle population. Recent experiments
suggest that these deleterious effects can be attributed to an acute loss of
vascular patency due to clot formation induced by damage to ovarian arterial
endothelial cells
STUDY DESIGN, SIZE, DURATION Study 1
(2010–2011; N = 16) examined the effect of post-thaw perfusion of survival
factors (angiogenic, antioxidant, anti-apoptotic; n = 7–8) and treatment with
aspirin (pre-operative versus pre- and post-operative (n = 7–9)) on the
restoration of ovarian function for 3 months after WOCP&TP. Study 2
(2011–2012; N = 16) examined the effect of cryoprotectant (CPA) perfusion
time (10 versus 60 min; n = 16) and pre- and post-operative treatment with
aspirin in combination with enoxaparine (Clexane®; n = 8) or
eptifibatide (Integrilin®; n = 8) on ovarian function and
fertility 11–23 months after WOCP&TP
PARTICIPANTS/MATERIALS, SETTING, METHODS Both studies
utilized mature, parous, Greyface ewes aged 3–6 years and weighing 50–75 kg.
Restoration of ovarian function was monitored by bi-weekly blood sampling and
display of behavioural oestrus. Blood samples were assayed for
gonadotrophins, progesterone, anti-Müllerian Hormone and inhibin A. Fertility
restoration in Study 2 was quantified by pregnancy rate after a 3 month
fertile mating period and was confirmed by ultrasound, hormonal monitoring
and live birth. Ovarian function was assessed at sacrifice by ovarian
appearance and vascular patency (Doppler ultrasound) and by follicular
histology
MAIN RESULTS AND THE ROLE OF CHANCE In Study 1,
survival factors were found to have no benefit, but the inclusion of
pre-operative aspirin resulted in four ewes showing acute restoration of
ovarian function within 3 weeks and a further six ewes showing partial
restoration. The addition of post-operative aspirin alone had no clear
benefit. In Study 2, combination of aspirin with additional post-operative
anti-coagulants resulted in total acute restoration of ovarian function in
14/14 ewes within 3 weeks of WOCP&TP, with 9/14 ewes becoming pregnant
and 4/14 giving birth to a total of seven normal lambs. There was no
difference between anti-coagulants in terms of restoration of reproductive
function and fertility. In contrast, the duration of CPA perfusion was highly
significant with a 60 min perfusion resulting in ovaries of normal appearance
and function with high rates of primordial follicle survival (70%) and an
abundant blood supply, whereas ovaries perfused for 10 min had either
resorbed completely and were vestigial (7/14) or were markedly smaller (P
< 0.01). It is concluded that both the degree of CPA penetration and the
maintenance of post-operative vascular patency are critical determinants of
the success of WOCP&TP
LIMITATIONS, REASONS FOR CAUTION Before
application of this technology to fertility preservation patients, it will be
critical to optimize the CPA perfusion time for different sized human ovaries,
determine the optimum period and level of anti-coagulant therapy, and confirm
the normality of offspring derived from this procedure
WIDER IMPLICATIONS OF THE FINDINGS This
technology holds promise for the preservation of fertility in women. It could
also potentially be applied to the cryopreservation of other reproductive or
even major organs (kidneys) where there are considerable difficulties in
storing donated tissue
STUDY FUNDING/COMPETING INTEREST(S) Funding was
received from the Medical Research Council, University of Nottingham. The
authors confirm that they have no conflict of interest in relation to this
work
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