Alexander Lopata

Vitrification can be more favorable than slow cooling

OBJECTIVE Cryopreservation of embryos and oocytes has become an essential service for infertility treatment. The clinical application of this technology should ensure optimal survival of the embryos and oocytes that are stored and subsequently thawed for transfer. The aim of this review is to compare the widely employed slow cooling procedures with vitrification to evaluate and recommend the more effective and safer procedure. DESIGN The review is mainly based on a comparison of the principles, procedures, and results reported in the literature. A historical description of vitrification and personal experiences with this technology are also included. SETTING University-based hospitals and private clinics that treat infertility and have published information on cryopreservation. PATIENT(S) Women being treated for infertility and reproductive technology clinics. INTERVENTION(S) The application of slow cooling involving a range of cooling rates is compared with vitrification using rapid and ultrarapid cooling in simple containers. The purpose of both techniques is the induction of a glasslike state in cells to protect them from damage by ice crystals. The early development of vitrification involved the use of long pre-equilibration procedures. Improved methods resulted from the use of mixtures of penetrating and nonpenetrating solutes that are not toxic and a range of cooling rates. MAIN OUTCOME MEASURE(S) Reported number of pregnancies established after transfer of embryos that were cryopreserved by vitrification, or transfer of embryos derived from vitrified oocytes. RESULT(S) Both slow cooling and vitrification procedures have resulted in the successful cryopreservation of human embryos and oocytes. Both procedures have resulted in healthy births, although the slow cooling of oocytes gives very low success rates. Vitrification is a promising novel technique in assisted reproductive technology, but comparative success rates are yet to be established. CONCLUSION(S) Vitrification is a simple procedure that requires less time and is likely to become safer and more cost effective than slow cooling.

Concepts in human in vitro fertilization and embryo transfer

The significantly higher fertilization rate and embryo yield in 1981 compared with 1980 is likely to be due to two main factors. These include the larger average follicular diameter used for timing the hCG injection and the delayed insemination of preovulatory oocytes both of which were introduced in the second half of 1981. When the effects of each of these factors were examined separately it was found that delayed insemination did not significantly increase the fertilization rate of eggs derived from leading (largest) follicles. However it was found that when preovulatory eggs derived from secondary follicles were preincubated for up to 6 hours before insemination their fertilization rate was significantly greater than that of equivalent eggs inseminated immediately after recovery. It would appear therefore that the higher fertilization rate observed in 1981 was largely due to the delayed insemination which significantly enhanced the fertilizability of eggs derived from second-order follicles. This effect may be due to progressive oocyte maturation during the incubation interval. (authors)

A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro

A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetra-methylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.

Presence of active gelatinases in endometrial carcinoma and correlation of matrix metalloproteinase expression with increasing tumor grade and invasion

The actions of the extracellular‐matrix degrading enzymes, matrix metalloproteinases (MMPs), are implicated in tumorigenesis. The cellular localization of MMP‐2, MMP‐9, membrane type 1 (MT1)‐MMP, tissue inhibitors of metalloproteinases (TIMPs) 1‐3, and the presence of active gelatinases were investigated in endometrial carcinoma.

Analysis of the benefits and risks of multiple embryo transfer.

Depending on embryo quality and recipient factors, increasing the number of embryos transferred after in vitro fertilization may produce more pregnant patients or more multiple pregnancies. An analysis is outlined demonstrating how observations on the placement of two embryos in the uterus may be used to estimate the risks and benefits associated with the transfer of multiple embryos. Currently, the highest pregnancy rates are obtained with multiple embryo transfers, and these results are compared with the mathematical model.

A sperm-zona pellucida binding test and in vitro fertilization.

Sperm binding to the zona pellucida was studied in 106 in vitro fertilization (IVF) patients. Oocytes that failed to fertilize in vitro were inseminated with a mixture of equal numbers of test and fertile donor sperm differentially labeled with fluorescein or rhodamine to control for variability in the sperm-zona pellucida binding capacity of oocytes. The ratio of the number of test and control sperm bound to four to six zonae pellucidae was significantly correlated with sperm morphology, viability, motility, motility index, and normal intact acrosomes in semen. The sperm-zona pellucida binding ratio was the most significant factor related to IVF rates by logistic regression analysis. But the proportions of sperm with normal morphology and intact acrosomes in semen also were significant. In patients with

Preovulatory follicular size: a comparison of ultrasound and laparoscopic measurements.

Ovarian follicles in the immediate preovulatory period were measured with real-time or static ultrasound. Thirty-nine follicles were examined in thirty-six patients. In 29 spontaneous cycles the mean follicular diameter was 21.1 mm (range 17 to 25 mm) and the mean volume was 5.1 ml. In seven stimulated cycles the mean diameter was 18.4 mm and the mean volume was 3.5 ml. Laparoscopic needle aspiration of the follicular contents was performed within 12 hours of ultrasound examination in every case. Follicular dimensions based on the volume of aspirated fluid correlated well with the ultrasound measurements (r = 0.847; P < 0.001), which suggests that ultrasound is a useful technique for examining preovulatory follicular development.

Serum supplement in human in vitro fertilization and embryo development

The effects of maternal preovulatory serum and human fetal cord serum supplement in culture medium in human in vitro fertilization and embryo development were compared in 208 cases over an 8-month period when there were no significant changes in other variables. A significantly higher pregnancy rate was observed in the fetal cord serum group despite no significant difference being found in fertilization and embryo cleavage rates. This suggests a difference in the health of the embryo cultured in different serum supplements.

Uterine cavity matrix metalloproteinases and cytokines in patients with leiomyoma, adenomyosis or endometrial polyp.

OBJECTIVE To determine whether leiomyoma, adenomyosis and endometrial polyps are associated with changes in uterine cavity matrix metalloproteinases (MMP-2 and MMP-9) and cytokines. STUDY DESIGN Uterine cavity irrigation was performed in women with leiomyoma, adenomyosis and endometrial polyps, and in women with a normal uterus. MMP-2 and MMP-9 were assayed in the uterine washings by gelatin zymography. For individual subjects, the total MMP level was obtained by adding the semi-quantitative scores of band densities related to gelatinases in the zymograms. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) were measured using enzyme-linked immunosorbant assay (ELISA) kits. RESULTS The uterine cavity of patients with leiomyoma, adenomyosis and endometrial polyps had significantly higher MMP scores than controls. Although the mean IL-1beta levels were elevated in uteri harboring a pathology compared with the normal uteri, the cytokine was significantly elevated only in the adenomyotic group. Significantly elevated levels of IFN-gamma were found in uteri with leiomyoma and endometrial polyps. Uterine washings from leiomyoma and adenomyosis contained significantly elevated mean levels of TGF-beta1 compared with controls, while TNF-alpha was significantly higher only in leiomyoma. When uterine cytokine levels were compared in relation to individual MMP levels a significant relationship was found between TGF-beta1 and elevated levels of MMP-9 and total MMPs in leiomyoma. A significant relationship was also found between IL-1beta and elevated levels of MMP-2, MMP-9 and total MMPs in the endometrial polyp group. CONCLUSION The uterine cavity in leiomyoma, adenomyosis and endometrial polyps contains elevated levels of MMPs and cytokines compared with the normal uterus. In some pathologies elevated cytokines are associated with elevated MMPs.

Implantation in the marmoset monkey: Expansion of the early implantation site

This study was initiated to examine the early stages of trophoblast adhesion and invasion during implantation in the marmoset. Seven implantation sites were found in the uteri of four marmosets taken between days 13 and 15 of gestation. Three implantation sites in two uteri were examined in detail by electron microscopy. Between days 13 and 15, the marmoset implantation site expanded peripherally by adding areas where syncytial trophoblast penetrated between uterine luminal epithelial cells. Such penetrating masses often bridged openings of endometrial glands, shared junctional complexes with the uterine epithelial cells between which they are infiltrating, and subsequently reached the residual basal lamina of the uterine luminal epithelium. Centripetal to the peripheral region was an intermediate region in which syncytial trophoblast overlay individual clusters of epithelial cells and rested along the basal lamina. In this region there was some evidence of fusion of syncytial trophoblast with uterine epithelial cells. In the central region of the implantation site near the inner cell mass and amnion the trophoblast formed elaborate lamellipodia in relation to the basal lamina. In one of the three specimens examined with electron microscopy there were two foci where trophoblast penetrated through the basal lamina. It was also in the central region that trophoblast penetrated farthest into the uterine glands. The gland cells closest to trophoblast were less closely associated and lost their columnar shape, forming large round cells similar to the epithelial plaque cells of other primates. Where two blastocysts implanted on the same side of the uterus a conjoint membrane was formed which in regions consisted solely of syncytial trophoblast with two basal surfaces and two basal laminas. The prolonged period of time when the implantation site expands within the plane of the uterine epithelium (trophoblastic plate stage) and the peripheral to central sequence in extent of development make this primate a particularly useful animal for studies of trophoblast adhesion to and penetration of the uterine luminal epithelium. Anat Rec 256:279–299, 1999.