A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation program.

OBJECTIVE: To determine which factor, or factors, may influence the success of an embryo-freezing program. DESIGN: Prospective. SETTING: An established, private IVF clinic. PATIENTS: This study comprises data from 185 consecutive thaw cycles (161 patients) that resulted in embryo replacement. The influence of the following factors, with regard to pregnancy outcome, was specifically addressed: embryo quality, day of the endogenous LH surge, number of thawed embryos replaced, length of time frozen, E2 level at the time of the hCG trigger in the stimulated cycle, and patient age at the time of freezing. RESULTS: An overall pregnancy rate (PR) of 27.6% per ET was achieved in this study. Regardless of the day of the LH surge, patients who had at least one good quality embryo (the "sponsoring embryo") conceived 35% of the time. Patients who lacked a sponsoring embryo frozen conceived 15% of the time. Patients who were 39 years of age or younger had a PR of 31% per transfer. None of the 22 patients who were 40 or older conceived. No other factor examined had any effect on pregnancy outcome. CONCLUSIONS: Good quality frozen/thawed embryos are a major factor in achieving a consistently high PR. Poorer quality embryos are capable of implantation but at a significantly reduced rate. Patient age was also found to be significant in predicting pregnancy. Neither the day of the LH surge, number of embryos transferred, length of time frozen, nor the E2 level appears to have any effect on the establishment of pregnancy.

Ultrastructural observations of human and mouse oocytes treated with cryopreservatives.

The effects of the cryopreservative agents dimethylsulfoxide (DMSO) and propanediol (PROH) on mature human and mature mouse oocytes have been examined with transmission electron microscopy. Treatment of CD-1 mouse oocytes and human preovulatory oocytes in a stepwise manner with either DMSO or PROH up to 1.5 M appears to trigger the exocytosis of 70-80% of the cortical granules in all oocytes. Successive stages in premature dehiscence, including a loss in granule electron density, fusion of the granule-limiting membrane with the oolemma, and extrusion of the cortical granule core into the perivitelline space, have been observed in all human oocytes studied. In addition, all human DMSO- and PROH-treated oocytes exhibited crypt-like invaginations and clusters of endocytic vesicles that subtend the oolemma. The presence of these crypts and pinocytotic vesicles in treated oocytes may suggest a mechanism for the retrieval of cortical granule membrane that is inserted into the original plasmalemma during exocytosis. The paucity of cortical granules in treated mouse and human oocytes as it potentially relates to an impaired ability to elicit the cortical reaction at fertilization is discussed.

An ultrastructural analysis of an oocyte from an in vitro fertilization patient with repeated polyspermic fertilization.

OBJECTIVE: Our goal was to determine any ultrastructural anomalies in an oocyte from a patient with a history of polyspermy. RESULTS: Ultrastructural observations of the cortical ooplasm of several oocytes from each of three control patients showed a large population of intact cortical granules. Conversely, one oocyte from a patient with repeated polyspermic fertilization contained a relative paucity of granules in the cortex. Quantitative analysis of the cortices of control oocytes indicated that there were 17.02 +/- 0.52 cortical granules present per measured field of view, compared with 4.40 +/- 2.92 granules per field in the other oocyte. CONCLUSIONS: The presence of sufficient cortical granules is necessary for normal (monospermic) fertilization to occur. When contrasted to the cortical granule population of oocytes from several control patients, the cortex of one oocyte from the other patient showed few of these organelles. Therefore, the absence of a sufficient number of granules may have precluded normal fertilization from occurring in the eggs of this patient.

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.