The choice of a gonadotropin-releasing hormone analog influences outcome of in vitro fertilization treatment.

OBJECTIVE: Our purpose was to determine if there is a difference in outcome associated with choice of gonadotropin-releasing hormone analog in in vitro fertilization treatment cycles. STUDY DESIGN: A retrospective analysis of 510 consecutive in vitro fertilization cycles with patient-selected use of either nafarelin (Synarel) or leuprolide (Lupron) was performed. RESULTS: Of 510 consecutive patient cycles, 284 patients (56%) chose nafarelin and 226 (44%) chose leuprolide. In the nafarelin group 64 cycles (34% of retrievals) resulted in deliveries. In the leuprolide group 37 (24%) resulted in delivery (p < 0.05). There were 260 patients in their first cycle of treatment, with 157 (60%) choosing nafarelin, resulting in 33 deliveries (34% per retrieval). Leuprolide, used in 103 (40%) of first cycles, resulted in 12 deliveries (20% per retrieval), (p = 0.052). CONCLUSIONS: In a large population of unselected patients undergoing in vitro fertilization the choice of nafarelin was associated with a significantly better outcome in terms of successful pregnancies achieved.

The resazurin reduction test provides an assessment of sperm activity.

OBJECTIVE: The objective of the study was to determine if reduction of the dye resazurin by semen could be correlated with the concentration of motile sperm. DESIGN: After assessment of sperm count and motility, specimens were incubated for 1 hour with resazurin (25 micrograms/mL of semen) and visual color changes indicative of dye reduction noted. SETTING: Specimens were obtained from men seeking care for infertility at one of four sites: (1) University of California, San Francisco (UCSF) In Vitro Fertilization Program; (2) UCSF Andrology Laboratory; (3) a gynecological practice in Maine; and (4) a private andrology laboratory in Southern California. PATIENTS: Individuals were self-selected by their desire to have a semen analysis in conjunction with the diagnosis or treatment of infertility. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The reduction of the dye resazurin and its correlation with motile sperm density. RESULTS: When the motile sperm concentration was greater than or equal to 20 X 10(6)/mL, 86% of specimens produced a positive color change. Conversely, 86% of specimens with a motile sperm concentration of less than 20 X 10(6)/mL either did not change color or changed only over a narrow range. CONCLUSION: Reduction of resazurin offers an assessment of the active sperm in a specimen without the need to do a sperm count or evaluation of motility.

Maturation of immature oocytes by coculture with granulosa cells.

To increase the number of embryos available for transfer, immature human oocytes were cocultured with granulosa cells from preovulatory follicles. Greater numbers of immature oocytes incubated with granulosa cells had dispersion of the cumulus and corona cells compared with immature oocytes cultured in media alone. Fifty-four percent of immature oocytes were fertilized after coculture with granulosa cells compared with 20% fertilization of immature oocytes cultured without granulosa cells. There were no cases in which only embryos developed from immature oocytes were transferred, and thus we could not determine if the immature oocytes could contribute to a pregnancy.

Development of two-cell mouse embryos in protein-free and protein-supplemented media.

Two-cell mouse embryos from CFW (Swiss-Webster) mice were cultured to the blastocyst stage in Ham's F10, Whittingham's T6, or human tubal fluid medium. Media were used without any protein supplements or were supplemented with human maternal serum, human fetal cord serum, or human serum albumin. Blastocysts were transferred to modified Eagle's basal medium supplemented with 10% fetal bovine serum for postblastocyst development. Blastocyst and postblastocyst development was depressed among embryos cultured during the preimplantation stage in protein-free Whittingham's T6 and human tubal fluid media compared with embryos cultured in protein-free Ham's F10 medium. This advantage of Ham's F10 disappeared when amino acids and vitamins were added to the other two media. Whittingham's T6 and human tubal fluid supplemented with human serum albumin, human maternal serum, or human fetal cord serum also supported excellent embryo development. When supplemented with protein, Ham's F10 was the poorest of the media in supporting embryo development. Although these results suggest that Ham's F10 is not the best medium for culture of mouse embryos, there is need for caution in extrapolating results from the mouse to the human.

Polypronuclear embryos after in vitro fertilization.

Triploidy occurs in 1% to 3% of recognized conceptions in vivo. In vitro fertilization (IVF), which places large numbers of sperm in proximity to the oocyte, may increase the risk of triploidy. The risk also could be influenced by stimulation protocols that differentially affect egg quality. We found no significant difference in triploidy when cycles were stimulated with clomiphene citrate/human menopausal gonadotropin (3.2%) compared with gonadotropin-releasing hormone analog/human menopausal gonadotropin stimulation (4.4%). Triploidy was less common with male factor infertility (0.7%) compared with other causes of infertility (3.4% to 3.8%). Triploid embryos can develop to cleavage stages indistinguishable from those generated by diploid embryos. Thus, identifying and discarding embryos with greater than 2 pronuclei is a vital function of an IVF laboratory.

Polyovular follicles associated with human in vitro fertilization.

Polyovular follicles were found in 61 (24%) of 251 laparoscopies performed for in vitro fertilization. Of 898 follicles that contained at least one oocyte, 76 (8%) were polyovular. When the oocytes from polyovular follicles were assessed by morphologic criteria, it was found that 46 of the follicles contained oocytes that were discordant in maturity. Thus, oocytes can develop at different rates even though they are exposed to the same follicular fluid. It is possible that the microenvironment provided by the cellular investments of oocytes may correlate more precisely with oocyte maturity.

Development of mouse embryos in vitro is affected by strain and culture medium.

One-cell and two-cell embryos from three random-bred strains of mice--CF1, Dub:(ICR), and CFW (Swiss-Webster)--were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.

The effect of nitrous oxide on in vitro fertilization success rate.

The authors studied the effect of nitrous oxide on success rates for in vitro fertilization and pregnancy in women undergoing laparoscopy for oocyte retrieval. Ninety-eight patients in an in vitro fertilization program were randomly assigned to an anesthetic regimen including either 0.7% (end-tidal) isoflurane with 60% nitrous oxide in oxygen, or 1.4% (end-tidal) isoflurane in oxygen. Success rates for fertilization and pregnancy in 44 additional patients who declined randomization were also studied. Among the 51 randomized patients who did not receive nitrous oxide, 192 oocytes were obtained and 122 fertilized (63.5%), resulting in eight pregnancies (16.3%). From the 47 randomized patients given nitrous oxide, 168 oocytes were retrieved and 114 fertilized (67.9%), resulting in nine pregnancies (19.1%). No significant differences between rates of fertilization or pregnancy emerged between groups. Such differences would have been found with an 80% probability had nitrous oxide had a 20% effect on oocyte fertilization.

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation.

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17beta-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or epsilon-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0

Private adoption.

During an 18-month period 219 couples and 3 single women were counseled concerning private, non-agency adoption. Ninety-seven clients adopted privately a total of 105 babies. Thirty-one other couples are still actively pursuing adoption, 39 are no longer pursuing adoption, 13 became pregnant, 9 adopted through an agency, and 33 were lost to follow-up. One hundred of the 105 adopted babies were placed with the family directly from the hospital at 2 to 3 days of age. The majority of the adopting parents had a baby in their home within 4 months of the time they actively began trying to adopt. Four babies were reclaimed by the biologic mother before the adoption was finalized in court, but three of the couples that lost their babies adopted again. The average cost of a private adoption in this series was $3300. Private adoption is an important alternative to agency adoption.

The free surface of mouse trophoblast in culture is non-adhesive for other cells.

Dispersed cells from cultured mouse cell lines, mouse macrophages, and inert microspheres were layered onto outgrowing mouse trophoblast in culture. The cells that settled onto the trophoblast remained round, in contrast to the elongated spreading shape they assumed on the glass substratum. The cells were readily dislodged from the trophoblast surface, whereas the microspheres were strongly adherent to trophoblast within 30 min. Scanning electron microscopy showed that trophoblast engulfed the spheres, but not the cells. Despite the lack of adhesion between cells and trophoblast, cell processes connected the two. The inability of cells to adhere to the free surface of the trophoblast could explain the trophoblast's ability to induce contact inhibition in co-cultured cells.

Spontaneous cure of male infertility.

Four of sixteen couples whose infertility was thought to be due to a male factor achieved a pregnancy without treatment. Pregnancy without therapy, despite compromised sperm counts or motilities, has been documented by a number of other authors. Despite this information many studies concerning the treatment of male infertility neglect to include control groups, and pregnancies which occur are credited solely to the therapy. Such claims of therapeutic success should be viewed with caution.

Intrauterine insemination of isolated motile sperm.

Semen from infertile males was filtered through liquid albumin, and sperm retrieved from the most distal fraction were used for intrauterine insemination. Compared with the ejaculate, the isolated specimens contained fewer sperm but an increased percentage of motile sperm. The fractions were also free of the debris (white cells, agglutinated sperm, immature forms, and particulate matter) commonly seen in the ejaculates. Despite the improvement in motility none of the 19 women who were treated for a total of 67 cycles became pregnant. It is possible that even the best sperm from a poor specimen may have intrinsic defects that interfere with their ability to fertilize.

PIP: An albumin column technique for isolating motile sperm from ejaculate was used to artificially inseminate 19 women, members of couples who suffered long-term infertility (4.5 years, range 1-10 years) thought to have a male factor as the primary cause. Semen from infertile males was filtered through liquid albumin, and sperm from the most distal end of the fraction were used for intrauterine insemination. Th isolated specimens contained fewer sperm but an increased percentage of motile sperm compared with the ejaculate. Women were treated for a total of 67 cycles (8 for only 1 or 2 cycles; none for more than 8 months). Though the sperm fraction isolated on albumin columns showed good motility, numbers apparently adequate for conception (as gleaned from previous animal and human artificial insemination studies), more uniform morphology, and absence of seminal debris, no pregnancies occurred during treatment cycles. Possibly even the best sperm from a poor specimen have intrinsic defects that interfere with their ability to fertilize, since previous reports of normal pregnancies following intrauterine insemination have been documented.

Sex preselection.

There have been attempts to select the sex of a child prior to conception in both animals and humans. Centrifugation, microelectrophoresis and density gradient sedimentation have been used to separate X and Y sperm in animals. Only the latter technic has produced a change in sex ratio. In the human, timing of coitus or artificial insemination in relation to ovulation has been the popular method for influencing the sex of a child. This review suggests caution in accepting the claims of success for coital timing. Sperm bearing the Y chromosome can now be identified by quinacrine staining. Using this marker, Ericsson showed that when sperm are allowed to swin into columns of liquid albumin a high percentage of Y-bearing sperm are found in the most distal portion of the column. At this time it has not been shown whether the fraction enriched with Y sperm can produce a preponderance of males.

Differential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured mouse embryos.

Treatment of preimplantation mouse embryos in vitro with 10(-3) to 10(-1) mug actinomycin D/ml for 2 hr showed that (i) postimplantation development in vitro was inhibited most when embryos were treated at the morula stage and (ii) after the morula stage actinomycin D inhibited trophoblast outgrowth less than inner cell mass development.