BACKGROUNDSuccessful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice.OBJECTIVE AND RATIONALEThe objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method.SEARCH METHODSA Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI.OUTCOMESOne RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05–7.51; P = 0.039; 48 and 30 cycles, respectively, per transfer (RR = 1.81, 95% CI 0.71–4.67; P = 0.214; 47 and 19 transfers) and per warmed/thawed oocyte (RR = 1.14, 95% CI: 1.02–1.28; P = 0.018; 260 and 238 oocytes). One RCT comparing vitrification versus fresh oocytes was analysed. In vitrification and fresh cycles, respectively, no evidence for a difference in ongoing CPR per randomized woman (RR = 1.03, 95% CI: 0.87–1.21; P = 0.744, 300 women in each group), per cycle (RR = 1.01, 95% CI: 0.86–1.18; P = 0.934; 267 versus 259 cycles) and per oocyte utilized (RR = 1.02, 95% CI: 0.82–1.26; P = 0.873; 3286 versus 3185 oocytes) was reported. Findings were consistent with relevant cohort studies.Of the seven RCTs on embryo cryopreservation identified, three met the inclusion criteria (638 warming/thawing cycles at cleavage and blastocyst stage), none of which involved pronuclear-stage embryos. A higher CPR per cycle was noted with embryo vitrification compared with slow-freezing, though this was of borderline statistical significance (RR = 1.89, 95% CI: 1.00–3.59; P = 0.051...
This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.
We sought to determine the influence of different constituents of the extracellular matrix on porcine granulosa cell function by assessing cellular attachment, cellular morphology, follicle-stimulating hormone (FSH) receptors, and progesterone production. Cells from immature porcine ovarian follicles were cultured for up to 6 days in serum-free medium containing porcine FSH (pFSH, 10 ng/ml) in culture dishes either uncoated or coated with one of the following adhesion proteins: gelatin (1 mg/cm2), fibronectin (1 microgram/cm2), laminin (1 microgram/cm2), type I collagen (10 micrograms/cm2), or type IV collagen (7.8 micrograms/cm2). Fibronectin, laminin, type I collagen, and type IV collagen increased cellular attachment significantly (P < 0.05). All adhesion proteins except gelatin influenced cellular morphology. Cells cultured on laminin or type IV collagen formed dense clusters of rounded cells. Cells cultured in dishes coated with each adhesion protein except gelatin had higher 125I-pFSH binding per cell than cells cultured in uncoated dishes, with increases of 7- to 12-fold over control (P < 0.05). All adhesion proteins increased progesterone production, ranging from 10- to 50-fold over control (P < 0.05). In summary, not only did adhesion proteins increase attachment to the dishes but they also increased FSH receptors and differentiated function (progesterone production) of granulosa cells from immature porcine ovarian follicles.
The goal of the present studies was to compare direct effects of porcine FSH (pFSH) on [125I]pFSH-binding sites with effects of pFSH on FSH receptor mRNA in cultured porcine granulosa cells. Cells from immature follicles were cultured on laminin-coated plates in serum-free medium for up to 6 days in the absence or presence of pFSH (1-100 ng/ml) or cholera toxin (0.04-400 ng/ml), which activates adenylyl cyclase independently of the FSH receptor. RRA indicated that [125I] pFSH binding to cells cultured without stimulator increased more than 10-fold with time in culture. Addition of pFSH to cultures resulted in a dose-dependent decrease in binding, assessed after removal of bound pFSH. Equilibrium saturation binding analysis indicated that pFSH (10 ng/ml) caused a 39% decrease in binding sites in cells cultured for 6 days. At the same time, pFSH increased progesterone production 9.5-fold. Cholera toxin (4 ng/ml) increased [125I]pFSH binding 110% and progesterone production 8.9-fold. Northern hybridization analysis of cultured granulosa cell mRNA using a porcine FSH receptor cDNA revealed three transcripts for the FSH receptor [2.2, 3.5, and 4.2 kilobases (kb)], with the major transcript being 4.2 kb in length. Addition of either pFSH (10 ng/ml) or cholera toxin (10 ng/ml) to cultures of granulosa cells increased the intensity of pFSH receptor transcripts compared with control values, with the 4.2-kb message remaining predominant. Hybridization with a porcine LH receptor cDNA revealed different transcripts (2.4, 4.0, 4.7, 7.0, and 11.0 kb), with the major transcript being 4.7 kb in length. Addition of either pFSH or cholera toxin to the cultures increased the intensity of all LH receptor transcripts; however, cholera toxin was more effective than pFSH. pFSH and cholera toxin increased the intensity of each species to different extents, although the 4.7-kb transcript remained predominant. These results indicate that exposure to FSH in culture results in down-regulation of the FSH receptor. Down-regulation is accompanied by increased FSH receptor mRNA levels, suggesting that FSH enhances FSH receptor synthesis.
Granulosa cells were aspirated from small follicles from the ovaries of 3- to 6-month-old pigs, washed, and cultured in sealed spinner flasks. Medium 199 was supplemented with 10% fetal bovine serum, insulin, dexamethasone, T4, and antibiotics. FSH, LH, or hCG was added daily where appropriate. Cultures were sampled every other day for 10 days. Cells were examined microscopically, and cellular [125I]hCG binding and DNA content were measured. The cells aggregated and underwent morphological luteinization within 4 days in the absence or presence of FSH, LH, or hCG. Aggregation in culture was independent of clumping at the time of harvest. There was no measurable cell proliferation, based on the lack of mitotic figures and the lack of any increase in DNA. The average yield of luteinized cells from small follicle granulosa cells was 7% at 6 days. Cell concentration remained constant between days 6-10. Between days 0-2, [125I]iodo-hCG binding declined in all cultures. It continued to decline through day 10 in control cultures. In cultures containing FSH, binding increased to a maximum on day 6. The effect was specific for FSH. Increased binding was due to increased numbers of binding sites per cell. Equilibrium saturation binding data indicated that [125I]iodo-hCG binding to freshly harvested and cultured granulosa cells was specific, saturable, and of high affinity. The numbers of binding sites were 938 +/- 49 and 7602 +/- 156 sites/cell for control and FSH on day 6, respectively, compared to 498 +/- 14 sites/cell on day 0. The effect of FSH on the increase in binding sites was specific and dose related. Autoradiographic studies indicated a skewed normal frequency distribution of [125I]iodo-hCG-binding sites on cultured cells
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