B. Salle

Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification

OBJECTIVEnTo evaluate recovery of endocrine function and fertility after transplantation and vitrification of whole ovaries.nnnDESIGNnAnimal study.nnnSETTINGnLyon Veterinary School, France.nnnANIMAL(S)nEwes.nnnINTERVENTION(S)nIn group 1 (n = 5), the left ovary was removed with its vascular pedicle and was transplanted onto the contralateral pedicle. In group 2 (n = 5), the left ovary with its pedicle was cryopreserved after a vitrification procedure. After thawing, transplantation was performed by microvascular anastomosis to the contralateral ovarian pedicle.nnnMAIN OUTCOME MEASURE(S)nMedian ischemia time, progesterone levels, histologic examination.nnnRESULT(S)nSuccessful microsurgical transplantation was performed in both groups. The median ischemia time was statistically significantly longer in group 2 (287 minutes, range: 226 to 349] versus 129 minutes [range: 125 to 130]) in group 1. In group 1, four sheep recovered spontaneous ovarian endocrine function about 2.5 (range: 2.00 to 3.75) months after transplantation. Two ewes gave healthy live births at 12 and 25 months, respectively, after transplantation. In group 2, one ewe recovered ovarian endocrine function 6 months after transplantation. However, histologic evaluation showed a follicular survival rate of 6% in group 1, and total follicle loss in group 2.nnnCONCLUSION(S)nAutograft of whole sheep ovaries with microvascular anastomosis seems technically feasible but resulted in a very poor follicle survival rate (6%), in spite of endocrine function recovery and birth of two lambs. Attempts at cryopreservation with vitrification resulted in no follicle survival at all.

Repeated ovarian stimulation with corifollitropin alfa in patients in a GnRH antagonist protocol: no concern for immunogenicity

BACKGROUND One injection of corifollitropin alfa replaces the first seven daily FSH injections in controlled ovarian stimulation (COS) cycles. Repeated treatment with therapeutic proteins may cause immune responses or hypersensitivity reactions. We assessed the immunogenicity and safety of corifollitropin alfa treatment in up to three COS cycles. METHODS In this multicentre, phase III uncontrolled trial, patients (>60 kg) started treatment with one injection of 150 µg corifollitropin alfa on cycle Day 2 or 3 of menses and 0.25 mg ganielix on stimulation Day 5 or 6. Primary outcome measures were antibody formation against corifollitropin alfa (using highly sensitive radioimmunoprecipitation assay), hypersensitivity reactions, local tolerance and adverse events (AEs). RESULTS First, second and third COS cycles were started by 682, 375 and 198 patients, respectively. No clinically relevant immunogenicity or drug-related hypersensitivity was observed. For 192 patients undergoing their third cycle a post-treatment blood sample was negative in the anti-corifollitropin antibody assay, resulting in an upper limit of the one-sided 95% confidence interval (CI) of 1.5%. Most frequent AEs were procedural pain (17.7%, 95% CI: 14.9–20.8%), headache (9.1%, 95% CI: 7.0–11.5%) and pelvic pain (7.6%, 95% CI: 5.7–9.9%). Cumulative ongoing pregnancy rate after three cycles, including frozen-thawed embryo transfer cycles and spontaneous pregnancies, was 61% (95% CI: 56–65%) after censoring for patients who discontinued. CONCLUSIONS Treatment with corifollitropin alfa can safely and effectively initiate and sustain ovarian stimulation during the first 7 days of COS in normal responder patients undergoing up to three treatment cycles, without concerns of immunogenicity. The trial was registered under ClinicalTrials.gov identifier NCT00696878.

Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe

BackgroundThe objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures.MethodsEwes ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests.ResultsOvarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p < 0.05) or calcein AM/ethidium homodimer-1 staining (r = 0.76, p < 0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60 +/- 1.1% vs 52.2 +/- 7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26 ± 8.2% vs 38 ± 4.5%).ConclusionWe suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

Validation of a new metabolic marker to assess the vascular viability of vitrified whole sheep ovaries

BACKGROUNDnWhole ovary cryopreservation has been suggested as a means to preserve fertility. In animal models, autologous cryopreserved ovary transplants frequently undergo thrombosis and a method to assess the vascular viability of cryopreserved ovaries would be valuable. We developed a staining method using methylthiazolyl blue tetrazolium (MTT, a metabolic marker) to assess the pedicle metabolism of whole ovaries vitrified using cryoprotectant called VS4.nnnMETHODSnWhole sheep ovaries were perfused with MTT (1 g/l). In one group, ovarian tissue lesions were induced by immersing the ovarian pedicle in medium at 53°C or 65°C or in liquid nitrogen prior to MTT perfusion. In the second group, several metabolic substrates (d-glucose, l-glucose and pyruvic acid) and inhibitors [2-deoxy-d-glucose for d-glucose metabolism, azide for mitochondrial respiration and diphenyleneiodonium (DPI) for NADPH oxidase (an effector of the pentose phosphate pathway)] were added to the MTT stain. The third group was subjected to VS4 ± vitrification/warming prior to MTT perfusion. Pedicle MTT staining was assessed qualitatively by histological examination of frozen sections or quantified at 564 nm after solubilization in alcohol.nnnRESULTSnMTT strongly and reproducibly stained the vascular smooth muscle. Heating at 53°C or 65°C or cooling in liquid nitrogen significantly diminished MTT staining by 48% (P = 0.001, n = 10), 94% (P = 0.0002, n = 10) and 94% (P = 0.0002, n = 10), respectively. MTT staining was affected by d-glucose metabolism: absence of d-glucose, substitution of unmetabolized l-glucose for d-glucose or addition of 2-deoxy-d-glucose significantly decreased MTT staining by 44% (P < 0.01, n = 10), 45% (P < 0.01, n = 10) and 29% (P < 0.01, n = 10), respectively. Pyruvic acid failed to correct the MTT staining decrease induced by d-glucose deprivation and azide did not decrease MTT staining, suggesting that MTT staining could be independent of mitochondrial metabolism. Adding DPI significantly inhibited MTT staining by 25% (P < 0.001, n = 10), suggesting involvement of the pentose phosphate pathways effectors. Compared with controls, VS4-vitrified/warmed pedicles showed significantly less MTT staining (-30%, P < 0.005, n = 10), with unstained foci, whereas unvitrified VS4-exposed pedicles showed no difference.nnnCONCLUSIONSnMTT can serve as a qualitative and quantitative vascular viability marker.VS4 vitrification caused alterations in ovarian vascular metabolism. MTT staining should allow accurate comparisons of whole-organ cryoprotection protocols.

Technical aspects of laparoscopic ovarian autograft in ewes after cryopreservation by slow-cooling protocol.

Iatrogenic ovarian failure and infertility are long term-term side effects of anticancerous gonadotoxic treatments in children or women of reproductive year. Ovarian cortex cryopreservation can be a solution to preserve immature germinal cells before gonadotoxic treatment, for later transplantation. The aim of our study was to prove the efficiency of a laparoscopic technique for orthotopic graft after a slow-freezing/thawing protocol, and to evaluate the effect of ovarian cryopreservation and autograft on the primordial follicle survival rate. Experimental surgical study was performed on 6- to 12-month-old ewes. The study was approved by the ethic committee of the Lyon-veterinary-school. The left ovary was removed by laparoscopy and cut in half, and medulla was excised. In group 1 (n = 6), autograft was performed immediately on the right ovary, and in group 2 (n = 6), graft was performed after a slow-freezing/thawing protocol. The second hemi-ovary served as an ungrafted control fragment. A polypropylene/polyglactin mesh was included between graft and base to separate the two structures, to help histological analysis. The mean graft performance time was 71 +/- 8 min in the first group and 57 +/- 10 min in the second. Freezing did not affect the number of primordial follicles. In the ungraft control fragments, the global anomaly rate (cytoplasm plus nuclear anomaly) increased after freezing (p < 0.05). Others results did not reach signification. Pelvic adhesion occurred only once. The post-graft primordial follicle survival rate was 5.1 +/- 2.8% in the non-frozen group vs. 6.3 +/- 2.3% after freezing/thawing. Kruskal-Wallis and Wilkoxon non-parametric tests were used for statistical analysis. Laparoscopy seems to be a well-adapted technique for ovarian tissue orthotopic autograft. The main follicle loss occurs before graft revascularization. Our orthotopic grafts procedure has to be improved to obtain a better grafts neovascularization, and to have a better long-term post-graft primordial follicle survival rate.

Factors related to unstained areas in whole ewe ovaries perfused with a metabolic marker

STUDY QUESTIONnWhat factors are associated with the presence of areas unexposed to the perfusate after whole ovary perfusion?nnnSUMMARY ANSWERnOver half the ovaries perfused with the metabolic marker methylthiazolyl blue tetrazolium (MTT) were incompletely stained. Incomplete staining was statistically significantly associated with a small ovarian slice surface area, inexperience of the experimenter, and the presence of a corpus luteum.nnnWHAT IS KNOWN ALREADYnWhole ovary cryopreservation followed by vascular auto-transplantation has provided poor outcomes as an alternative way to safeguard fertility. Perfusion, commonly used to expose the ovaries to cryoprotectants, may miss areas excluded from the vascular network, explaining subsequent poor ovarian functionality.nnnSTUDY DESIGN, SIZE, DURATIONnAn observational study of 360 ewe ovaries stained by in vitro perfusion with MTT as a qualitative marker of tissue blood supply was performed. A logistic regression model was built to identify factors associated with incomplete ovary staining.nnnMATERIALS, SETTING, METHODSnWhole ewe ovaries with their vascular pedicles were perfused at 0.35 ml/min with 1 g/l MTT for 2 h at 39°C under 19 experimental conditions. The pedicles were removed and the ovaries cut in half sagittally and photographed. The unstained area of the slice surface was measured. Times from ovary collection to ovary rinsing and to MTT perfusion initiation, ovary weight and slice surface area, presence of a corpus luteum and operator experience (number of ovaries previously perfused) were recorded. Pedicle MTT staining was quantified at 564 nm after solubilization in alcohol.nnnMAIN RESULTS AND THE ROLE OF CHANCEnUnstained areas were observed in 64.4% of the ovaries. Multivariate analysis found that incomplete ovary staining was independently associated with lower experimenter experience (P < 0.02), smaller ovary slice surface area (P < 0.0001) and presence of a corpus luteum (P < 0.01). The presence of unstained areas was independent from experimental conditions. The rate of incomplete ovary staining decreased from 83 to 60% beyond the 80th perfused ovary (P < 0.0001).nnnLIMITATIONS, REASONS FOR CAUTIONnDescriptive study.nnnWIDER IMPLICATIONS OF THE FINDINGSnBlood-supply impairments that result in incomplete perfusion might adversely affect outcomes after whole ovary cryopreservation. Improved perfusion techniques should enhance success.

La vitrification : technique d’avenir pour la cryoconservation ovarienne ? Bases physiques de cryobiologie, avantages et limites

Ovarian cryopreservation is presently indicated in patients who undergo a gonadotoxic treatment, most commonly for anticancer procedures. These procedures can strongly alter fertility by damaging the follicular ovarian reserve. Although six human live births have been described in the world after ovarian tissue cryopreservation and autografting, the techniques of cryopreservation techniques are not consensual. Vitrification is a physical process that allows cryopreservation without formation of ice crystals, by transformation of a highly concentrated solution in a glassy or amorphous state. Vitrification is at present rapidly expanding in the biology of reproduction. With the classic methods of freezing, formation of ice crystals within the ovarian tissue is systematic and can entail cellular lesions. Which is why more and more teams question the theoretical advantage of the vitrification for ovarian cryopreservation. Our objective was to summarize the fundamental physical basis of cryobiology, necessary for an understanding of vitrification. From our experience, we also wanted to point out the practical difficulties of this technique, and we are proposing a model of evaluation and validation that uses differential scanning calorimetry, applicable to any protocol of vitrification.

État actuel de la folliculogenèse in vitro chez la souris

Follicle culture systems have been developed so as to achieve in vitro fertilization of oocytes coming from immature follicles. The in vitro folliculogenesis methods would be especially useful in reproductive medicine to restore fertility in women having undergone ovarian cryopreservation. Several culture systems allowing in vitro growth of small follicles have been developed in mouse. These have proven to be successful by the birth of healthy offsprings. Some elements determine the outcome of culture: follicle isolations at a defined stage of development, follicular morphology preservation, and supplementation of growth factors or hormones. Development of follicle culture in the mouse model led to a better understanding of ovarian physiology, in particular the relation between endocrine and paracrine factors on follicle development. The in vitro techniques in mouse became a valuable tool for improving reproductive technics improvement, and for toxicology studies.

Acute efficacy of a sublingual dose of nifedipine on uterine arterial blood flow: preliminary data in prematurely menopausal women.

To determine whether the calcium blocker nifedipine alters Doppler velocimetry and impedance parameters in the uterine artery in prematurely menopausal women.

OC090: Acute efficacy of a sublingual dose of nifedipine on uterine arterial blood flow in premature menopausal women

In-vitro maturation is increasingly viewed as an alternative to IVF in selected patients. Its primary attractions are its reduced costs, reduced health risks and improved patient acceptability. Ultrasound has a pivotal role in both patient selection and monitoring in IVM. The number of immature oocytes retrieved correlates with the number of follicles present. Oocytes are retrieved from around 50% of follicles seen. Patients with polycystic ovaries by definition have multiple small follicles and therefore are ideal candidates for IVM. Patients with normal ovaries may be suitable depending on the number of follicles present in both ovaries. At the commencement of a cycle of IVM treatment, patients have a transvaginal ultrasound on day 2 to 4 of the cycle, primarily to count the number of follicles present which will allow an estimation of the number of oocytes likely to be recovered. A second scan is performed on day 8/day of hCG to assess for the presence of a dominant follicle and endometrial thickness. A cycle is usually cancelled if a dominant follicle is present over 10 mm on the day of hCG. The endometrial thickness on the day of oocyte retrieval will determine the dose of estradiol required for endometrial priming. Pregnancy rates are related to the number of immature oocytes collected and are approximately 30% if greater than 10 oocytes are collected.