Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation of whole adult sheep ovaries.

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 INTERESTS: 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.

Effects of dehydroepiandrosterone on in vivo ovine follicular development.

STUDY QUESTION: What are the effects of exposure of ovarian tissue to dehydroepiandrosterone (DHEA) supplementation in vivo? SUMMARY ANSWER: DHEA exposure stimulates initiation of primordial follicles and development of gonadotrophin-responsive preantral/early antral follicles possibly mediated through promoting granulosa cell proliferation and enhancing anti-Mullerian hormone (AMH) expression. WHAT IS KNOWN ALREADY?: Ovarian ageing is a cause of subfertility and is associated with poor outcomes of IVF treatment and premature menopause. A few clinical studies have shown that DHEA can improve ovarian response and increase the chances of pregnancy after IVF treatment in women with a diminished ovarian reserve (DOR) suggesting DHEA may help to overcome the effect of ovarian ageing. However, there are no data about how DHEA acts on ovarian folliculogenesis. STUDY DESIGN, SIZE AND DURATION: A cortical autograft experimental model was conducted in six female sheep aged at least 24 months. The period of DHEA treatment in the animals lasted for 10 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: All the animals were subjected to unilateral oophorectomy. Half of the ovary was fixed for histological analysis as a time-zero control. The remaining tissue was used to isolate patches of ovarian cortex which were autografted back onto the ovarian pedicle. The grafting procedure eradicated all growing follicles and synchronized early follicular development. After a 10-week treatment period with DHEA implants, the ewes were sacrificed and the graft and remaining ovary were harvested. Histological and immunohistochemistry (IHC) findings, accompanied with serum hormonal profiles were compared to determine the effect on the follicle population. MAIN RESULTS AND THE ROLE OF CHANCE: Higher proportions of the follicle population in the remaining ovary were observed to be in the antral stage after DHEA treatment. The observation coincided with an increase in the rate of primordial follicle initiation and preantral follicle development in cortical grafts and the remaining ovarian tissue, respectively. The IHC results indicated that DHEA increased the expression of both the proliferation marker (KI-67) in granulosa cells and the follicular AMH expression at the preantral and early antral follicle stages. LIMITATIONS, REASONS FOR CAUTION: The experimental design compared follicle populations before and after DHEA treatment within individual animals to allow changes over time to be detected against a background of high inter-animal variation. However, since no controls without DHEA were included, we cannot say what would have happened over time in its absence, and it is possible that other factors may have resulted in the changes in follicle development observed during the experiment. WIDER IMPLICATIONS OF THE FINDING: Our data supports the idea that DHEA might be a useful therapy to delay the effects of ovarian ageing. Therefore, it may have a role as an adjunct during IVF to improve ovarian response in women with DOR and as a treatment for premature ovarian insufficiency. STUDY FUNDING/COMPETING INTEREST(S): The research received finance support from the University of Nottingham. The authors declare no conflict of interest in this study.

Intracycle variation in number of antral follicles stratified by size and in endocrine markers of ovarian reserve in women with normal ovulatory menstrual cycles.

OBJECTIVE: To quantify the intracycle variation in markers of ovarian reserve measured by antral follicle counts stratified by size using three-dimensional (3D) ultrasound and anti-Müllerian hormone (AMH) in women with normal menstrual cycles. METHODS: Healthy volunteers with normal menstrual cycles were prospectively recruited. Three-dimensional (3D) ultrasound examination and blood test were performed in early (F1) and mid-follicular (F2) phases and in periovulatory (PO) and luteal (LU) phases of one menstrual cycle. Antral follicles were measured using 'sonography-based automated volume calculation' with post processing (SonoAVC) and ovarian volume was measured using Virtual Organ Computer-aided AnaLysis (VOCAL). Blood serum was processed for hormonal assays including AMH, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol. Repeated-measures analysis was used to examine the variance in markers of ovarian reserve in different phases of one menstrual cycle. RESULTS: A total of 36 volunteers were included in the final analysis, of whom 34 attended all four visits. Repeated-measures analysis showed a significant variation in total antral follicle count (AFC) (P < 0.001). However, on stratifying the antral follicles according to size using SonoAVC, a non-significant variation (P = 0.382) was seen in small AFC (≤ 6.0 mm) and a significant variation (P < 0.001) was seen in large AFC (> 6.0 mm). The ovarian volume showed a significant intracycle variation (P < 0.001). A small but significant intracycle variation was noted in AMH (P = 0.041) and a significant variation was seen in levels of serum FSH, LH and estradiol (P < 0.05). CONCLUSION: Small antral follicles (≤ 6.0 mm) measured using 3D ultrasound and AMH show little intracycle variation and perhaps should be evaluated when predicting ovarian reserve independent of menstrual cycle.

The effects of whole ovarian perfusion and cryopreservation on endothelial cell-related gene expression in the ovarian medulla and pedicle.

Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature. This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in the follicle population and ovarian function in vivo following short-term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial cell-related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial, transitional and primary follicle densities were significantly reduced (P < 0.05) and stromal Ki67 and caspase-3 expression significantly increased (P < 0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere (FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilizing a customized RT-PCR array or conventional RT-PCR, we found that perfusion alone resulted in down-regulation in the expression of caspase 6 and thrombospondin 1 (THBS1) genes in the medulla. Following additional cryopreservation, endothelial nitric oxide synthase (eNOS), endothelin 1, endothelin receptor A and Bcl-2 expression were significantly (P < 0.05) down-regulated. In the pedicle, both perfusion and cryopreservation caused a (P < 0.05) down-regulation of eNOS and THBS1, and an up-regulation in Bax expression. Perfusion also caused a down-regulation of TNF and up-regulation of endothelin-2 expression (P < 0.05). In conclusion, this study has identified a number of endothelial cell-related genes expressed in the medulla which are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious effects on endothelial cell function.

Quantifying effect of combined oral contraceptive pill on functional ovarian reserve as measured by serum anti-Müllerian hormone and small antral follicle count using three-dimensional ultrasound.

OBJECTIVES: Oral contraceptive pills suppress the hypothalomo-pituitary axis, which can affect the ultrasound and endocrine markers used to examine ovarian reserve. The objective of this study was to quantify the ultrasound and endocrine markers of functional ovarian reserve in women using a combined oral contraceptive pill (COCP) for more than a year. METHODS: This was a prospective case-control study involving healthy volunteers: 34 women using for more than a year a COCP with hormone-free interval (HFI) were compared to 36 normo-ovulatory, age-matched controls who had not used hormonal contraception within the last year. Volunteers using a COCP underwent a 3D ultrasound examination and had a blood sample taken within the first 4 days of active pill ingestion and those in the control group had the scan and blood test in the early follicular phase (days 2-5) of menstrual cycle. The main outcome measure was the difference in antral follicle counts stratified according to size and anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels. RESULTS: There were no significant differences in the number of small antral follicles measuring 2-6 mm. The COCP group had significantly fewer antral follicles measuring ≥ 6 mm (P < 0.001) and had significantly smaller ovaries (P < 0.001), which also had lower vascular indices than the control group (P < 0.05). While serum FSH, LH and E2 levels were significantly lower in the COCP group (P < 0.05), there was no significant difference in serum AMH levels between the two groups. CONCLUSIONS: Prolonged use of COCP suppressed pituitary gonadotropins and antral follicle development beyond 6 mm, but had no effect on levels of serum AMH and number of small antral follicles.