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

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.

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.

Development of the mouse blastocyst after actinomycin D treatment.

Mouse blastocysts were incubated in vitro with actinomycin D in concentrations of 10-1 to 10-5 mug/ml. After transfer to recipient animals, there was a moderate but significant decrease in implantations at 10-minus 1 to 10-minus 3 and 10-minus 5. There was a severe inhibition of subsequent development at concentrations of 10-minus 1 and 10-minus 2. One of 49 fetuses which developed after actinomycin D treatment had a major malformation; there was one major and one minor abnormality among 95 control fetuses. It is suggested that most of the RNA or protein required to effect implantation is synthesized or coded for prior to the blastocyst state. The moderate but significant decrease in implantations found after actinomycin D treatment at this stage may reflect the ability of the drug to reduce cell adhesiveness. This study confirms the impression that the preimplantation embryo is relatively impervious to teratogenic agents, even one as potent as actinomycin D.

Effects of pH on the migration of X and Y sperm.

PIP: A study was undertaken to examine the influence of pH on the migration of X and Y sperm as observed in freshly ejaculated semen samples from human donors. These sperm were allowed to migrate into capillary tubes containing media of varying pH. Y-carrying sperm were i dentified using flourescent staining with quinacrine dihydrochloride. S mall changes in pH were ineffective in influencing the ratio of sperm. 6 additional samples were analyzed in a more acid pH resulting in an insignificant change in migration. This study indicated that the percentage of Y-bearing sperm migrating through a capillary tube is unaffected by the medium's pH. Evidence suggest the unlikelihood that X and Y sperm can be differentiated on the basis of migration through media of varying pH.^ieng

Sperm-agglutinating antibodies in infertile women.

PIP: This paper explored the experience with sperm-agglutininating antibodies in 168 infertile couples. 2 of the 46 subjects with organic causes for infertility had positive sperm agglutination tests, and neither became pregnant in the 6-18 month followup period. Of the remaining 122 subjects with no organic cause for infertility, 24 (20%) had positive tests (including 13 women with primary infertility and 11 with secondary infertility). 9 of the 24 women (38%) with positive tests became pregnant following condom therapy. There was no correlation between the sperm antibody test and postcoital test, nor between sperm agglutinins and ABO incompatibility. These results suggest that a small number of women have specific reactions against sperm or seminal fluid that inhibit fertility. It is concluded that screening of serum for sperm agglutinins should be continued to provide an estimate of immunologic reactions against semen in cases of infertility.^ieng

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.

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.

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.

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.

Pregnancy following hysterosalpingography with oil and water soluble dye.

PIP: To determine whether hysterosalpingography (HSG) increases the conception rate and to compare the therapeutic effectiveness of oil and water soluble contrast media, the histories of 744 women who attended the Yale Infertility Clinic in 1965-69 were reviewed. Exclusion of women who had been infertile less than 1 year before coming to the clinic, in whom a HSG was done outside the clinic, and in whom there was no follow-up resulted in a study population of 460. The pregnancy rate for the study group (49%) was identical to that for the larger population. The study group was divided into women who became pregnant during their clinic attendance or within 1 year of terminating clinic care and women who did not become pregnant in this period. These 2 categories were then subdivided according to whether a HSG was done. To determine the relative effectiveness of different media, the conception rate following HSG in the Yale Clinic, where the iodized oil Ethiodol was used, was compared with that among 63 women who had HSG done by a private practitioner with the water soluble dye Salpix. 221 women were in the Ethiodol HSG group, 121 (55%) of whom conceived. Of the 239 Yale Clinic patients who did not have HSG, 103 (43%) became pregnant. Exclusion of couples with organic factors that could account for infertility resulted in a pregnancy rate of 58% for the HSG group and 47% for the non-HSG group. 25 (40%) of the 63 women in the Salpix group conceived, but exclusion of couples with organic factors lowered the pregnancy rate to 38% in this group. The average length of infertility was 0.5 years longer in the non-HSG group than in the Ethiodol HSG group, but shorter in the Salpix HSG group compared to the Ethiodol group. These results suggest that Ethiodol HSG may enhance fertility. Although the oil media has been criticized on the basis that it may cause granuloma formation or embolization, documented complications in the Yale series were rare. In contrast to earlier studies, unilateral nonpatency on x-ray was not found to affect eventual conception (58% pregnancy rate among women in the Ethiodol HSG group with normal tubes compared with 50% in those with filling of only 1 tube).^ieng

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.