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

STUDY QUESTION: What factors are associated with the presence of areas unexposed to the perfusate after whole ovary perfusion? SUMMARY ANSWER: Over 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. WHAT IS KNOWN ALREADY: Whole 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. STUDY DESIGN, SIZE, DURATION: An 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. MATERIALS, SETTING, METHODS: Whole 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. MAIN RESULTS AND THE ROLE OF CHANCE: Unstained 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). LIMITATIONS, REASONS FOR CAUTION: Descriptive study. WIDER IMPLICATIONS OF THE FINDINGS: Blood-supply impairments that result in incomplete perfusion might adversely affect outcomes after whole ovary cryopreservation. Improved perfusion techniques should enhance success.

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

BACKGROUND: Whole 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'. METHODS: Whole 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. RESULTS: MTT 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 pathway's 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. CONCLUSIONS: MTT 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 graft's procedure has to be improved to obtain a better graft's neovascularization, and to have a better long-term post-graft primordial follicle survival rate.

Prospective evaluation of the threat related to the use of seminal fractions from hepatitis C virus-infected men in assisted reproductive techniques.

BACKGROUND: The risk of hepatitis C virus (HCV) transmission during assisted reproductive techniques (ARTs) is still disputed and no report concerning its prospective evaluation is available. METHODS: The aim of this 4-year follow-up multicentre study that enrolled 86 HCV-serodiscordant couples was to determine whether a sperm-processing method was able to reduce levels of HCV in semen and the risk of HCV transmission to the newborn. All the men were chronically infected by HCV and 10 of them by human immunodeficiency virus. A total of 181 seminal plasmas and 153 sperm fractions were tested for the presence of HCV RNA. RESULTS: HCV RNA tested positive in 20.4% of the seminal samples. All of the 153 final sperm fractions tested negative for HCV. The detection of HCV RNA in semen was significantly correlated with a high viral load in blood (P < 0.05). The presence of HCV RNA in seminal plasma impaired neither semen parameters nor ART issue. From the 58 couples enrolled effectively in an ART programme, 24 pregnancies and 28 newborns were obtained. All of them tested negative for HCV RNA in blood. CONCLUSION: These results emphasize the safety of the semen-processing method. The negligible risk of transmitting HCV reduces the value of the systematic analysis of HCV RNA in seminal fractions prior to ART. Since use of this analytical procedure involves the freezing of semen, its avoidance would result in an increase in sperm quality and reduce the need to perform intracytoplasmic sperm injection techniques.

Dynamic CpG methylation of the KCNQ1OT1 gene during maturation of human oocytes.

BACKGROUND: Imprinted genes, many of which are involved in development, are marked during gametogenesis to allow their parent-of-origin specific expression, and DNA methylation at CpG islands is part of this epigenetic mark. Maternal imprint is apposed on oocyte during growth and maturation. Factors interfering with normal oocyte differentiation such as gonadotrophin stimulation and in vitro maturation (IVM) may possibly alter imprint resetting. METHODS: We examined the methylation of the KCNQ1OT1 differentially methylated region (KvDMR1) in human oocytes at different stages of their development: germinal vesicle (GV), metaphase I (MI) or metaphase II (MII). RESULTS: About 60% of alleles were fully methylated in GV oocytes and that full imprint is acquired in most MII oocytes. Similarly to in vivo, de novo methylation of DNA occurred in vitro during oocyte maturation. Following in vitro culture for 28 h, GV and MI oocytes are significantly more methylated when they are obtained from natural cycles than from patients undergoing gonadotrophin stimulation. CONCLUSION: This observation suggests that hyperstimulation likely recruits young follicles that are unable to acquire imprint at KvDMR1 during the course of the maturing process.

[Vitrification: a future technique for ovarian cryopreservation? Physical basis of cryobiology, advantages and limits]. / 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.

[State of the art on in vitro folliculogenesis in mouse]. / Etat 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.

The impact of histones linked to sperm chromatin on embryo development and ART outcome.

The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone-to-protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c-IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = -0.12, p < 0.05). Regarding the type of ART procedure (c-IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR<6%; p < 0.01) and 74.6% (HPR >26%; p < 0.01). The highest delivery rate (DR; 24.5%) was obtained for HPR within the range [6%; 26%]; DR was 21.9% for HPR<6% and 18.3% for HPR>26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy.

Towards whole sheep ovary cryopreservation.

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 degrees C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12-16 degrees C/min from -196 to -133 degrees C) in liquid nitrogen vapour, followed by rapid thawing in a 45 degrees C water bath at about 200 degrees C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4+/-2.2% of small follicles were viable while histological analysis showed that 48+/-3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.

[Ovarian tissue vitrification: Cortex and whole ovary in sheep]. / Vitrification du tissu ovarien: cortex et ovaire entier chez la brebis.

OBJECTIVE: The aim of this study was to evaluate a cryopreservation technique by vitrification of cortex or whole ovaries in sheep, using two cryoprotectant solutions: VS1 and VS4 and to study their physical properties to avoid ice crystallisation by vitrification of whole sheep ovaries permeated with a cryoprotectant solution. ANIMALS AND METHODS: From 6-month-old ewes, whole sheep ovaries with their vascular pedicles were collected at the slaughterhouse or at the veterinary school and prepared for cryoprotectant toxicity tests and freezing procedure. Follicle viability was measured by trypan blue test and histological examination of ovary. The hemi-ovarian cortex was stored in liquid nitrogen. Four to six weeks after the first laparotomy, the controlateral ovary was removed and the vitrified-warmed hemi-ovary was sutured. Thermal properties of a cryoprotectant solution called VS4 (critical cooling rates [Vccr], vitreous transition temperature [Tg], end of melting temperature [Tm]) were measured by differential scanning calorimetry. RESULTS: No significant difference in follicle viability or normal follicle rates was observed between ovarian cortex exposed or non-exposed to cryoprotectant solutions. Nor was any significant difference observed before and after vitrification. Three pregnancies occurred, from which four lambs were born after autografts of vitrified ovarian cortex. With whole ovary, the decrease in the number of normal follicles was lower when frozen-thawed ovaries were treated with VS4 (P = 0.04). There were less nuclear anomalies (P = 0.02). The Vccr of VS4 has been estimated to be 14.3+/-1.1 degrees C/min and Tg was -125.0+/-0.2 degrees C. Because the penetration of cryoprotectants was very low, Vccr was very high and the cooling speed did not allow cortex to vitrify. DISCUSSION AND CONCLUSIONS: Cryopreservation of cortex or whole ovary by vitrification seems a promising technique in reproductive medicine. The best histologic results were obtained with the VS4 cryoprotectant when whole ovary was vitrified.

[In vitro maturation: the French experience]. / Maturation ovocytaire in vitro: l'expérience française.

In vitro oocyte maturation (IVM) allows the use of immature oocytes in IVF. IVM does not require ovarian stimulation and therefore can be offered to patients at risk of ovarian hyperstimulation syndrome. IVM can be offered as an alternative to conventional IVF in women with PCOS (polycystic ovarian syndrome). However, cleavage, implantation, and pregnancy rates were lower than those obtained with conventional IVF. The present study describes the results of IVM in French IVF departments.

[Follicular morphology after ovarian cortex cryopreservation in the rabbit doe(Oryctolagus cuniculus)]. / Morphologie folliculaire après cryoconservation du cortex ovarien chez la lapine (Oryctolagus cuniculus).

OBJECTIVE: To compare the effects of two cryoprotective agents (DMSO and 1,2-PROH) used at two concentrations (1,5 and 2 M) on the morphology of small ovarian cortex follicles in doe. MATERIALS AND METHODS: Ovarian cortexes (n=40) were frozen in TCM199+10% FCS medium added to 1.5 or 2 M of DMSO or 1,2-PROH. Two controls were realized (fresh and frozen without cryoprotectant). The equilibration in cryoprotective solutions before freezing, and the elimination of the cryoprotective agents after thawing, was performed step by step. The effects induced by cryopreservation were evaluated by histological examination. RESULTS: Fresh ovarian tissue showed 68.6% of intact follicles. After freezing, only 1.5 M of 1,2-PROH preserved 48.0% of normal follicles, with no significant difference compared to the fresh control. The proportion of follicles without morphological defect observed after cryopreservation with DMSO was significantly reduced (respectively 28.8 and 34.8% for 1,5 and 2 M of DMSO). DISCUSSION AND CONCLUSIONS: Our results suggest that 1,2-PROH is a more effective cryoprotectant than DMSO, for the cryopreservation of doe ovarian cortex. These results differ from those that were obtained for other species, credibly because of a higher fragility of the ovarian tissue of the doe. Nevertheless, this species is an interesting animal model which allows rapid results after cryopreserved ovarian tissue graft.

[Administering GnRH Agonists in the luteal phase of Artificial Cycle Frozen-Thawed Embryo Transfers. A prospective randomized study]. / Administration d'agonistes de la GnRH (Gonadotrophin Releasing Hormone) en phase lutéale des protocoles substitutifs de transferts d'embryons congelés: étude prospective randomisée.

OBJECTIVE: Adding GnRH agonists in the luteal phase has recently been said to improve implantation in IVF treatment (increased rates of pregnancy and birth). Adding GnRH agonists could also be beneficial for frozen-thawed embryo transfers. The objective was to compare the administration of Gonadotropin Releasing Hormone (GnRH) agonists during implantation with usual progesterone supplementation in the artificial cycle of frozen-thawed embryo transfers. METHODS: A prospective randomized controlled trial was conducted in a reproductive medicine center in a university hospital including all women starting an artificial cycle of Frozen-Thawed Embryo Transfers (FET). Two hundred and twenty women were randomized from September 2013 to June 2014. In the addition of GnRh agonists' group, two triptorelin injections of 0.1mg were carried out on the 4th day and on the 6th day following the introduction of progesterone. The primary outcome was the ongoing pregnancy rate. RESULTS: The ongoing pregnancy rate was higher (17 % versus 10.6 % P=0.29) when triptorelin was added, although the difference wasn't significant for the population as a whole. The increase proved to be significant in the case of day 2 embryos (34.6 % versus 10.3 % P<0.05) and of vitrified blastocysts (33.3% versus 12.5% P<0.05). CONCLUSION: The ongoing pregnancy rate for day 2 embryos and vitrified blastocysts significantly increased when GnRH agonists were added during implantation.

[Predictive value of vitamin D plasma levels on pregnancy rates in a cohort of IVF patients]. / Valeur prédictive du dosage de vitamine D sur les taux de grossesse dans une cohorte de patientes suivies en FIV.

OBJECTIVE: To determine a possible correlation between plasma levels of vitamin D and pregnancy rates obtained by in vitro fertilization (IVF). PATIENTS AND METHODS: One hundred and ninety-eight womens participated in an IVF cycle from January to May 2012 in a prospective study. During the follicular phase locking, serum fluid was collected for vitamin D, calcium, FSH and estradiol analysis. The serum bhCG was checked 16 days after oocyte collect. Clinical pregnancy was confirmed by transvaginal sonography with at least one gestational sac in the uterine cavity. RESULTS: The mean levels of vitamin D was 31.7 nmol/L. A total of 169 patients (85.3%) had a vitamin D insufficiency (< 50 nmol/L). Only 29 patients (14.7%) had a sufficient vitamin D status (vitamin D 50 nmol/L). Pregnancy rate was 29.8% (59/198). There was no significant correlation between the levels of vitamin D and mean age (P = 0.92), BMI (P = 0.16) and etiology of infertility (P = 0.78). In contrast, the levels of vitamin D mean were significantly lower in patients from North Africa (P < 0.0001) and Black African (P = 0.0003) compared to Caucasian patients. DISCUSSION AND CONCLUSION: No correlation was found between serum vitamin D level and the pregnancy rate in IVF cycle.

Motility and fertilizing capacity of epididymal human spermatozoa in normal and pathological cases.

OBJECTIVE: To investigate different factors involved in the outcome of in vitro fertilization (IVF) with epididymal spermatozoa in cases of vas deferens agenesis, with particular emphasis on sperm movement parameters. DESIGN: Prospective study in which sperm movement characteristics, level of puncture, and sperm preparation technique were studied relative to the outcome of IVF. SETTING: Département de Gynécologie, Oncologie Gynécologique, Sénologie, Médecine de la Reproduction, Hôpital Edouard-Herriot, Lyon France. PATIENTS: Thirteen couples underwent 15 IVF cycles. In 14 cycles (12 patients) epididymal spermatozoa could be recovered by surgical punctures. Epididymides were taken from 10 subjects in state of cerebral death to study epididymal spermatozoa under physiological status of the epididymis. RESULTS: The cleavage rate in IVF was low (25/158: 15.8%). In seven cases, embryo transfer was possible, leading to two twin pregnancies (4 infants born). Fertilization could be achieved with spermatozoa from the epididymis caput. Motility was paradoxically higher in the proximal epididymis than in the distal epididymis. In healthy epididymides, spermatozoa collected from the caput did not exhibit any progressive motility, whereas progressive motility was always present in the epididymis corpus. CONCLUSIONS: Our study showed that forward motility could be observed in the epididymis caput in patients with congenital absence of the vas deferens and that fertilization and ongoing pregnancies could be obtained from caput spermatozoa. On the other hand, in physiological status of the epididymis, forward motility was observed only as from the corpus area, confirming observations in other species.

Comparison of two blastocyst culture systems: coculture on Vero cells and sequential media.

OBJECTIVE: To compare two blastocyst culture systems: culture on Vero cells and sequential media in the same time period. DESIGN: Retrospective study. SETTING: Institutional practice assisted reproductive technology center. PATIENT(S): Ninety-nine selected patients undergoing IVF with blastocyst culture. INTERVENTION(S): In vitro fertilization and transfer of at least one expanded blastocyst after culture using either Vero cells or sequential media (S(2) medium). MAIN OUTCOME MEASURE(S): Blastocyst, pregnancy, and implantation rates. RESULT(S): The blastocyst formation rate (41.6% vs. 31.6%) is significantly increased with sequential media compared with the coculture system, but there is no statistically significant difference in the pregnancy and implantation rates (39.5% vs. 38.6% and 23.7% vs. 15.1%, respectively). CONCLUSION(S): Blastocyst culture on sequential media offers a better dynamic of embryo development (22% vs. 13.6% blastocyst at day 5). There was no statistically significant difference between the two groups in the take-home baby rate (18 vs. 12).

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols.

OBJECTIVE: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN: Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING: Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S): Lambs 5 to 6 months of age. INTERVENTION(S): Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. MAIN OUTCOME MEASURE(S): Follicular mortality and histologic structure. RESULT(S): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Freezing, thawing, and autograft of ovarian fragments in sheep: preliminary experiments and histologic assessment.

OBJECTIVE: To evaluate the effects of freezing and thawing on the histologic changes in ovarian fragments from sheep and to determine the feasibility of ovarian autografts. DESIGN: Histologic evaluation of follicles that survived after freezing at -196 degrees C for 2 weeks. Histologic evaluation of ovarian fragments 6 months after the autografts. SETTING: Laboratoire de Zootechnie, Ecole National Véterinaire, Marcy l'Etoile, France. ANIMAL(S): Six ewes aged 6 months to 1 year. INTERVENTION(S): Cortical fragments were prepared from the right ovary of 6 lambs and were grafted immediately to the contralateral ovarian hilus or were cooled slowly to -196 degrees C in medium containing dimethyl sulfoxide for 2 weeks. The autografts were recovered 6 months later. RESULT(S): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles. CONCLUSION(S): Freezing of ovarian fragments is possible without damaging the follicles. Ovarian autografts showed good recovery of ovarian structure.

Detection of cytomegalovirus in semen from a population of men seeking infertility evaluation.

OBJECTIVE: To determine the incidence of cytomegalovirus in the ejaculates of infertile men who were seropositive for IgG antibodies to cytomegalovirus. DESIGN: Prospective study. PATIENT(S): We tested cytomegalovirus infection in the semen of men participating in an IVF-ET program. MAIN OUTCOME MEASURE(S): IgG and IgM antibodies to cytomegalovirus were measured in sera. We used polymerase chain reaction (PCR) and cell culture to look for both cytomegalovirus DNA and infectious virus in the semen of 70 men with cytomegalovirus-specific antibodies detected in sera. RESULT(S): Of the infertile couples, 13.5% exhibited "mismatching" serology (i.e., detection of IgG antibodies to cytomegalovirus in male serum only and not in female serum) and constituted a potential risk for cytomegalovirus transmission. Cytomegalovrius was identified in the semen of two patients who were positive for IgG antibodies to cytomegalovirus. Cytomegalovirus DNA also was detected in one positive sample after centrifugation through a three-layer Percoll gradient. CONCLUSION(S): Human cytomegalovirus was present in the semen from a population of infertile men. Rapid detection can be achieved by molecular techniques such as PCR combined with a hybridization assay. Even though cytomegalovirus was infrequently detected in semen, these data must be considered in determining the risk of transmission and developmental anomalies in infected fetuses.

Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep.

OBJECTIVE: To evaluate the effects of freezing, thawing, and autograft of a hemi-ovary on steroid secretion, endometrial maturation, and ovarian histology in ewes. DESIGN: Experimental animal study. SETTING: Laboratoire de Zootechnie, Ecole Nationale Vétérinaire, Marcy l'Etoile, France. ANIMAL(S): Six lambs aged 6 months to 1 year old. INTERVENTION(S): Hemi-ovaries were prepared and frozen from the right ovary of six lambs and autografted 4 weeks later to the contralateral ovarian hilus. The autografts and the uterus were recovered 1 year later. Blood tests were performed each week to measure P concentration. MAIN OUTCOME MEASURE(S): Number of primordial follicles; levels of plasma P. RESULT(S): Histologic examination of ovarian slices after freezing showed no destruction of primordial, primary, secondary, or cavitary follicles. The frozen ovarian autograft showed good recovery of the macroscopic and microscopic ovarian structure. After autografting, histologic examination revealed primordial to cavitary follicles. Secretion of P started to rise 4 weeks after the autograft. Histologic analysis of the endometrium showed numerous glands, vessels, and mucous secretion. CONCLUSION(S): Frozen ovarian autografts achieved P secretion and endometrial maturity.