Maha M. Mahadevan

The relationship of tubal blockage, infertility of unknown cause, suspected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer

The success of in vitro fertilization (IVF) and embryo transfer has been examined with regard to five categories of infertility over a 2-year period. Fertilization rates in vitro were highest in women with bilateral tubal blockage and women treated for endometriosis. There was a significant reduction of approximately 13% in the fertilization rate of couples with idiopathic infertility and women who had failed to conceive after 12 cycles of artificial insemination by donor. A further substantial reduction in the fertilization rate occurred when the husband had low quality semen, particularly when no abnormality was detected in the wife. Repeated IVF in couples with idiopathic infertility eventually resulted in fertilization. It is recommended that donor spermatozoa not be used for cases of idiopathic infertility, but it may be needed in cases of poor semen quality. There were no differences in the pregnancy rates following embryo replacement in any of the groups studied, nor was there any detectable effect of age on fertilization or pregnancy rates up to the age of 44 years.

Effect of Cryoprotective Media and Dilution Methods on the Preservation of Human Spermatozoa

Der Einfluß von kryprotektiven Medien und Verdünnungsmedien auf die Erhaltung menschlicher Spermatozoen

Relationship of fine structure of sperm head to fertility of frozen human semen

Damage to the plasma membrane and acrosome of human spermatozoa was quantitated following dilution, freezing, and thawing. Semen was obtained from 23 donors of known fertility attending an artificial insemination program. Dilution of semen with the cryoprotective medium containing glycerol significantly altered all the fine-structural and semen parameters studied. Membranes of the sperm head were severely altered after freezing and thawing. The percentage of spermatozoa with intact plasma membrane, with intact acrosomes, or with intact or swollen acrosomes was significantly reduced in frozen/thawed semen, compared with fresh semen samples. The percentage of spermatozoa with an intact plasma membrane and acrosome was significantly and positively correlated with the fertility of frozen donors semen used for insemination (r = 0.6, P less than 0.001). The deleterious effects observed in the sperm head membranes following dilution and freezing and thawing may explain why generally the fertility of frozen/thawed semen is lower than that of fresh semen and may also explain individual variation in fertility of donors after insemination of frozen semen.

Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa

Summary: Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10°C per minute from room temperature (RT) to 5° C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (< 5° C/min). The optimum cooling rate from RT to 5° C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1° C per minute when cooled from RT to 5° C and subsequently frozen‐thawed in liquid nitrogen (LN2). The optimal freezing rate of 10° C per minute, from 5° to‐80°C, resulted in higher survival of human spermatozoa than slower (1.1° C/min) or faster (87.1° C/min) freezing rates. Slow thawing in 20 or 35° C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at ‐30° C or lower when compared with samples transferred at ‐15° C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at ‐80° C or lower. Transfer of human semen from LN2 to ‐25 to ‐30° C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196° C or ‐80 to ‐85° C. No significant differences were found between storage temperatures of ‐80 to ‐85° C and ‐196° C in the maintenance of spermatozoa viability for up to 90 days.

Purified human early pregnancy factor from preimplantation embryo possesses immunosuppressive properties

This study was undertaken to determine whether early pregnancy factor secreted by preimplantation embryos has immunosuppressive properties. Human early pregnancy factor was purified from embryo growth media of in vitro fertilized ova with ion-exchange and gel filtration chromatography. During each step of purification the fractions were tested for (1) early pregnancy factor activity with the rosette inhibition assay, (2) immunosuppressive properties with a concanavalin A-stimulated lymphocyte proliferation assay, and (3) purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that (1) human early pregnancy factor has a basic molecular weight of 14 kd, (2) early pregnancy factor has immunosuppressive activity, (3) polymers of early pregnancy factor also appear to be present in the embryo growth media, and (4) immunosuppressive factors other than early pregnancy factor are also secreted by preimplantation human embryos. Early pregnancy factor and other factor(s) produced by the preimplantation embryo may play a role in suppressing maternal cellular immune responses, thereby preventing maternal rejection of the embryo.

Effect of oocyte quality and sperm characteristics on the number of spermatozoa bound to the zona pellucida of human oocytes inseminated in vitro

Sperm characteristics and oocyte quality may play a role in in vitro fertilization. The objective of this paper is to analyze the effect of the quality of oocytes, the husbands semen characteristics, and category of the couples infertility on the number of spermatozoa bound to the zona pellucida. One hundred eightyone oocytes which failed to fertilize or failed to cleave were fixed in 2.5% glutaraldehyde 40 to 60 hr after insemination in vitro and examined under interference microscopy and the number of sperm bound to the zone pellucida was determined. The means ± SD of sperm bound to mature, immature, and atretic oocytes were 51.0±50.7, 7.3±12.1 10.4±7.8, respectively. Fertilized mature oocytes (81.0±53.3) had a significantly higher number of sperm bound to zonae compared to unfertilized oocytes (41.8±47.3). It is concluded that the number of sperm bound to zonae is functionally important. The sperm motility and the number of motile sperm used to inseminate oocytes were significantly correlated with the number of sperm bound to zonae, whereas sperm morphology and sperm concentration did not correlate. This study supports the notion that sperm motility is the single most important factor influencing fertilization of human oocytes in vitro.

Immunosuppressive activity in human in vitro fertilization (IVF) culture supernatants and prediction of the outcome of embryo transfer: a multicenter trial

The supernatants from cultured human oocytes fertilized in vitro contain low molecular weight factors that can suppress or stimulate the proliferative response of lymphocytes in vitro. The inhibitory and stimulatory effects are nonspecific and may be detected using cultured human or murine tumor cell lines. Using such a bioassay, we previously tested fetal cord serum-supplemented culture supernatant and found that an absence of suppression was correlated with an absence of subsequent pregnancy. To test this association further, additional samples were obtained from four different in vitro fertilization (IVF) units and studied blindly without knowledge of the pregnancy outcome. In this series, samples were obtained after the first 12–24 hr of sperm-oocyte incubation and all of the supernatants were from individual embryo cultures. The average number of preembryos transferred to those achieving pregnancy did not differ significantly from the number transferred to those not achieving pregnancy but the level of suppression was greater (8.7±1.9%) in those becoming pregnant compared to those not achieving pregnancy (0.8±1.5%). Twenty-two of 61 patients who received at least one embryo with a suppressive supernatant achieved pregnancy, whereas 0 of 19 patients received embryos lacking suppressive supernatants became pregnant. Two patients who received a single embryo from cultures with suppression became pregnant. Several problems with the bioassay method were defined. The culture medium in this series was always supplemented with adult serum, usually from the patient herself, and this serum could be suppressive. Also, cultures containing sperm were suppressive to a greater extent than could be explained by the serum. High-performance liquid chromatographic (HPLC) analysis indicated that the suppressive effect of the serum was related to molecules greater than 100,000 kd and that sperm supernatants contained additional activity in this size range. Pooled supernatants from patients with suppression achieving pregnancy confirmed the presence of two low molecular weight species of approximately 3700 and 1200, and these moieties were absent in those patients with suppressive supernatants who failed to become pregnant. Similar low molecular weight molecules were also present in supernatants from sperm alone, suggesting sperm as a possible origin. The data indicate that the presence of certain types of molecules in human IVF supernatants may predict an increased likelihood of implantation and subsequent pregnancy, whereas a deficiency of these molecules predicts failure. A more rapid assay to detect specifically the presence or absence of these molecules in supernatants from the first 24 hr of human IVF culture could be useful in sparing patients the transfer of embryos unlikely to succeed and in selecting embryos with an optimal chance of success.

Effects of low-dose human chorionic gonadotropin on corpus luteum function after embryo transfer

This paper reports the effect of low-dose human chorionic gonadotropin (hCG) on the levels of serum hCG, progesterone, and estradiol, luteal-phase length, and conception in 20 patients undergoing in vitro fertilization and embryo transfer (IVF-ET). Alternate patients in a group of 20 received 1000 IU hCG on the day of embryo transfer and 3 days after. Six and 9 days from embryo transfer 2000 IU hCG was given. The remaining patients served as controls. No patients in the treated group and four in the control group became pregnant. The endocrine profiles with respect to hCG, progesterone, and estradiol levels were similar in the treated patients compared with pregnant patients in the control group. Treated patients had significantly longer (18.0±1.1 days) luteal phases compared with nonpregnant patients in the control group (12.5±1.2 days), indicating that low-dose hCG prolonged the life of the corpus luteum. It was concluded that while the administration of low-dose hCG prolonged the life of the corpus luteum, it did not apparently improve the conception rate.

Growth of mouse embryos in bicarbonate media buffered by carbon dioxide, hepes, or phosphate.

The purpose of this study was to determine if mouse embryos could be grown successfully in a culture medium devoid of the carbon dioxide phase (CO2). Mouse embryos fertilized in vivo were collected and cultured in Hepes medium with and without bicarbonate (HCO3−) and a phosphate medium with and without HCO3−. In these experiments no CO2 gas phase was used. Further embryos were cultured in Whittinghams modified Tyrodes (T6) medium with a CO2 gas phase and served as controls. The degree of embryonic development was noted. Surviving blastocysts were transferred to the uteri of pseudopregnant mice and delivery at term was allowed to occur. There was no significant difference in the degree of embryonic development in those embryos cultured in T6 or Hepes medium (+HCO3−) or in the number of live offspring obtained when these blastocysts were placed within the mouse uterus. Although embryonic development apparently proceeded successfully in the phosphate (+HCO3−) medium, none of these blastocysts survived when transferred to mouse uteri. No embryonic growth occurred in either the Hepes or phosphate media which were devoid of HCO3−. It appears that a Hepes medium containing HCO3−, which uses no CO2 gas phase, is as effective as T6 medium, which uses a gas phase, in supporting in vitro mouse embryonic growth.

Galectin-3 Is a Substrate for Prostate Specific Antigen (PSA) in Human Seminal Plasma

Galectin‐3 is a multivalent carbohydrate‐binding protein involved in cell adhesion, cell cycle control, immunomodulation, and cancer progression, including prostate cancer. Galectin‐3 function is regulated by proteolytic cleavage that destroys galectin‐3 multivalency while preserving carbohydrate‐binding activity. In human semen, galectin‐3 is present in seminal plasma and is also associated with prostasomes, exosome‐like vesicles secreted by the prostate. In the current study, we characterized the proteolytic activity that cleaves galectin‐3 in human seminal plasma.