Sally Catt

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 μm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.

Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.

Effects of lamb age, hormone stimulation and response to hormone stimulation on the yield and in vitro developmental competence of prepubertal lamb oocytes

Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (<20, 20-50 and >100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3-4-week-old lambs only (P < 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3-4-week-old lambs (15.2-25.6%; P > 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6-7-week-old lambs (P < 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3-4-week-old lambs, although by 6-7 weeks of age a high response to stimulation reduces blastocyst formation.

Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation.

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100  ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30  min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0-93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro , and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 +/- 2.8 blastocysts per lamb.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

The influence of sample volume applied to the Makler sperm counting chamber upon the measured concentration of latex beads: A multi- centre study

Abstract Objective To undertake a multi-centre study to maximize the number of Makler chambers used. Methods A total of 15 laboratories participated with 31 Makler chambers. A suspension of latex beads was prepared to a concentration of 20 millions per milliliter, and 0.5 mL aliquots distributed to each participating laboratory. They measured the concentration on their Makler chamber(s) used for routine semen analysis by adding 3, 4, 5, 7 and 10 μL volumes of bead suspension to the chamber. Results There was no difference in within-chamber analysis of the bead concentration according to the volume of bead suspension applied within the range of 3–10 μL ( F 4,14 =2.634, P =0.056). However, the between-chamber effects were significantly different (F 30,124 =4.937, P =0.000), and 24/31 (77.5%) chambers tested had an average bias>10% compared to the target bead concentration. Conclusions A volume of 3–10 μL added to Makler counting chambers does not influence the concentration measured of latex beads, but the between-chamber variability and positive bias seen would suggest that other sources of error are present which are yet to be identified.

EFFECT OF CHEMICALLY DEFINED LIPID MOLECULES ON BLASTOCYST DEVELOPMENT AND OUTGROWTH

Address for Correspondence: Dr. Anagha Nawal, Assistant Professor, Dept. of Anatomy, Mahatma Gandhi Medical College, Aurangabad [MS], India. E-Mail: dranagha2000@gmail.com Introduction: Fatty acids play a diverse role in early embryonic development and implantation. This study examined the effect of a chemically defined additive containing saturated and unsaturated fatty acids on mouse fertilization and embryo development in vitro. Objective: To observe the effect of a chemically defined addition of fatty acids on in-vitro fertilization, early embryonic development and blastocyst outgrowth Study Design: F1 mice (4-6 weeks) were superovulated and 16 hours later oocytes were collected and prepared for IVF using spermatozoa from F1 males. Oocytes (n=355) were randomly allocated to global fertilization medium with (+) or without (-) chemically defined lipid (#11905-031, Life Global Technologies). Fertilization was checked (2-PN) 6-9hrs after insemination and embryos were grown up in blastocyst culture medium + lipid additive or controls. Embryo development was monitored daily until D5 when blastocysts were transferred to 96well plates. Blastocyst outgrowths were imaged on D6, D7 and D9 using XY-Cyclone software. Data was analyzed (Chi-squares) to compare the effect of lipid on fertilization rate, blastocyst development and outgrowths. Result: Oocytes (n=355) randomly allocated to control (n=170) or +lipid groups for fertilization showed no difference in fertilization rates in medium with (n=155) or without lipid. Addition of lipid to blastocyst medium resulted in a significant increase (p<0.05) in blastocyst formation (73% vs 87%). but produced no difference in blastocyst hatching or outgrowth area. Conclusion: A chemically defined lipid additive promoted embryonic development in vitro. Further studies are needed to examine the dose-response effect of lipid on fertilization and embryo development.

407. Effects of vitrification and slow-cooling on the structure of lamb and mouse ovarian tissues

Follicular dynamics of ovarian tissue varies with species and age. The morphology and distribution of primordial, primary and secondary follicles were examined using mouse and lamb ovarian tissue before and after cryopreservation. Ovarian cortical tissue was processed from mouse (C57BL6J × CBA) and abattoir-sourced lamb ovaries and sliced into fragments (1 mm3). The fragments were randomly divided into three experimental groups. For vitrification, the tissue fragments were equilibrated (10% v/v ethylene glycol (EG) and DMSO, 20 min) and transferred to vitrification solution (17% v/v e.g. and DMSO and 0.75M sucrose, 3 min), and loaded onto a Fibreplug (CVM kit, Cryologic). For slow-cooling (SC), up to 10 ovarian fragments were placed in slow cooling solution (10% v/v DMSO and 0.1M sucrose, 5 min, RT), and then loaded in straws before placed in a programmable freezer (Cryologic CL8800i). For histological comparison, fresh and cryopreserved-thawed ovarian tissues were fixed, embedded and sectioned (5µm) and stained with haematoxylin and eosin. Primordial, primary and secondary follicles were evaluated and the normality of follicular structures was scored. Differences between treatment groups were examined using a Chi Square test. In lamb, 83% of follicles assessed were primordial, 16.4% primary and 0.6% secondary whereas in mouse the ratios were 60%, 27% and 13% respectively. In lamb, proportion of follicles with normal morphology after thawing for vitrification (77.2%) and SC (79.3%) was not significantly different from fresh controls (83.1%). Similarly, in mouse, the proportion of normal follicles after warming for vitrification (92.3%) and SC (90.5%) was not significantly different from fresh controls (91.4%). No obvious detrimental effects on morphology of follicles derived from either vitrification or slow-cooling of lamb and mouse ovarian tissue was observed.