Elevated ovarian follicular fluid stem cell factor concentrations are associated with improved pregnancy rates in in-vitro fertilization cycles.

OBJECTIVE: To determine whether ovarian follicular fluid (FF) stem cell factor concentrations are associated with successful IVF pregnancies. DESIGN: Nested case-control design evaluation of stem cell factor levels from the FF of oocytes fertilized and transferred after controlled ovarian hyperstimulation. SETTING: University-based ART program. PATIENT(S): Infertile women undergoing IVF in a university-based ART program. INTERVENTION(S): Fifty-seven FF samples from a cohort of patients (n = 13) with tubal factor and unexplained infertility were stored at -80 degrees C and subsequently evaluated for stem cell factor concentration. Patients with endometriosis, polycystic ovary disease, and male factor infertility were excluded. Stem cell factor concentrations were measured using a commercially available ELISA kit according to the manufacturer's specifications. The groups were analyzed using a one-way analysis of variance, and significance was determined using the chi2 analysis of contingency table, the unpaired t-test, or the Mann-Whitney rank-sum test. MAIN OUTCOME MEASURE(S): FF stem cell factor concentration, pregnancy. RESULT(S): Stem cell factor concentrations were significantly higher in the FF of the patients who achieved successful pregnancies than in those who did not (641.7+/-75.2 pg/mL versus 475.5+/-50.58 pg/mL). CONCLUSION(S): Elevated FF stem cell factor concentrations are associated with an increased likelihood of IVF success. Therefore, stem cell factor may play a role in human follicular and oocyte development, and increasing infrafollicular stem cell factor concentrations may improve pregnancy rates after oocyte retrieval, fertilization, and ET.

Elevated serum progesterone levels on the day of human chorionic gonadotropin administration do not predict outcome in assisted reproduction cycles.

OBJECTIVE: To determine if the level of serum P drawn on the day of hCG administration predicts assisted reproductive technology (ART) outcome in patients undergoing stimulation with hMG under GnRH agonist (GnRH-a) suppression. DESIGN: Retrospective P assay of stored serum. PATIENTS: One hundred seventy-one patients (189 cycles) who had undergone GnRH-a suppression (leuprolide acetate or nafarelin) and stimulation with hMG for an ART procedure. MAIN OUTCOME MEASURES: Progesterone RIA of serum obtained on the day of hCG administration. Measurement of sequential serum LH values by RIA in those patients with the highest P levels. RESULTS: Pregnancy rates per oocyte retrieval were not correlated with the P level before hCG administration. There were 18 of 54 (33.3%) clinical pregnancies in those cycles with P < 0.9 ng/mL (conversion factor to SI unit, 3.180) and 42 of 135 (31.1%) clinical pregnancies in cycles with a P > or = 0.9 ng/mL. Significantly higher serum E2 levels and numbers of of follicles and oocytes obtained were observed in the high P cycles. There were no differences in the number of oocytes fertilized, the number of embryos transferred, or the implantation rate. However, a significantly higher percentage of mature oocytes were fertilized in the low P cycles (73%), as compared with the high P cycles (60%). CONCLUSIONS: Serum P levels before hCG administration do not predict the outcome of ART cycles in patients suppressed with GnRH-a before hMG stimulation. Lower fertilization rates observed in the high P cycles did not have an effect on clinical outcome.

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.

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.

Optimizing the sperm penetration assay with human follicular fluid.

Major limitations of the conventional sperm penetration assay are the inability to assess several aspects of sperm function (zona binding and penetration) and the absence of human ovulatory products known to influence fertilization. We therefore modified the sperm penetration assay by the addition of human follicular fluid (FF) to induce the acrosome reaction in an attempt to improve the false-negative rate of the conventional technique. In 26 patients with negative results, results became positive in 20 with human FF and the acrosome reaction increased fourfold. In 19 different men, we compared the results of the conventional and modified assays with outcome of in vitro fertilization. The false-positive rate was the same, but the false-negative rate was reduced from 40% to 7% with the addition of human FF.

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.

Laboratory setup for human in vitro fertilization.

None of the procedures utilized in human IVF are unique or technically very difficult to perform. However, when considering the nature of the material being handled, it is essential that all laboratory procedures, equipment, and supplies are optimal for IVF . The individuals handling the gametes and embryos must be suitably trained and experienced in handling mammalian gametes and embryos before working with human tissues. In addition, the technologists must be trained in proper tissue culture techniques. Above and beyond the training and experience of the technologist, it is necessary that all laboratory equipment and chemicals for medium preparation be of the highest quality and known consistency, and that periodic checks of the culture conditions be performed using the bioassay of mouse embryos. The functioning of the equipment must be constantly monitored and backup systems be in place wherever possible in order to minimize the chances that laboratory accidents will result in loss or damage of the embryos. Meticulous attention to all aspects of the laboratory procedures will result in the patient's having the best possible chance to obtain a pregnancy.

Characterization of long-term monolayer cultures of human granulosa cells from follicles of different size and exposed in vivo to clomiphene citrate and hCG.

To determine the effects of prolonged hCG treatment in vitro upon granulosa cells from follicles of various sizes previously exposed to clomiphene citrate and hCG in vivo, progesterone and relaxin concentrations of spent media were correlated with light microscopic and ultrastructural characteristics. Intact, freshly dispersed cells were characterized by numerous lipid droplets, elliptical mitochondria with tubular or lamellar cristae, moderate rough-surfaced endoplasmic reticulum (RER), sparse smooth-surfaced endoplasmic reticulum (SER), and few Golgi. After 10-24 days in culture, 2 morphologically distinct cell types, 'granulosa-type' and 'luteal-type', were noted at the light microscopic level. Ultrastructurally, lipid droplets decreased in number, mitochondria became pleomorphic, RER became more prominent and dilated, and Golgi became more widely dispersed. Tubular SER became abundant and annular nexuses became more numerous after hCG treatment in vitro. Granulosa cells generated from all follicles responded to hCG treatment with significantly increased progesterone secretion after 4 days in culture. Relaxin was not detectable in any sample of medium. This study shows that human granulosa cells from 15-25-mm follicles retain their differentiated function of progesterone secretion in long-term culture and recover responsiveness to hCG in vitro, as demonstrated by enhanced progesterone secretion and development of prominent SER and increased annular nexuses.

Follicular size and number in human in vitro fertilization.

In an attempt to maximize the success of in vitro fertilization (IVF) and embryo transfer (ET) as a treatment for human infertility, we have examined the relationship of follicular size and number to the rates of oocyte recovery, fertilization, cleavage, and ET in clomiphene citrate-stimulated cycles. The recovery of oocytes from follicles less than 20 mm in diameter was significantly reduced over that from larger follicles, and those oocytes that were obtained from smaller follicles showed a significantly lower rate of fertilization and cleavage. In addition, the overall chance that a patient would undergo ET was greater in a cycle in which more than one follicle 20 mm or larger was developing than in a cycle in which a single large follicle was developing. This latter observation suggests that attempts at laparoscopic oocyte retrieval should be confined to cycles in which more than one accessible large follicle is developing, thereby maximizing the success rate while minimizing the risk and expense for the patient.

Identification of phosphates on the membranes of guinea pig sperm.

Guinea pig epididymal sperm, incubated for ATPases at pH 7.0 or pH 9.0, localize reaction product on both the periacrosomal segment of the plasmalemma and the outer acrosome membrane. In other species, e.g., rabbit, Ca++-ATPase is identified with the outer acrosome membrane. It may transport Ca++ into the acrosome for activation of enzymes released during the acrosome reaction. The neutral ATPase is demonstrable on the periacrosomal plasmalemma and possibly modifies Ca++ concentration in the fluid around the acrosome. In guinea pig sperm, Ca++-ATPase is sensitive to centrifugation or washing of sperm which indicates that the ductal fluid has unusual properties for preservation of the acrosome. Inhibition of the enzyme by these treatments suggests that conditions on the plasmalemmal surface affect the acrosome membrane. Inability to separate reaction product on the plasmalemma from that on the acrosome membrane may be due to migration of reaction product across the periacrosomal space. However, the ATPases are elicited in the guinea pig under the same conditions as in other species. The pH 9.0 enzyme requires Ca++ while the enzyme at pH 7.0 has no ion specificities. Demonstration of these enzymes indicates that mechanisms of acrosome activation, similar to those in other sperm, are relevant to the guinea pig.

Electron microscope assessment of fertilization of rabbit ova treated with concanavalin A and wheat germ agglutinin.

Zonaless rabbit ova, exposed to Concanavalin A or Wheat Germ Agglutinin, then to uterine capacitated sperm produce pronuclear, 2 and 4 stage embryos that are indistinguishable from controls. Absence of cortical granules indicates that the ova were fertilized and not merely activated. Survival of lectin-bearing receptors during the period necessary for fertilization was evaluated in ova marked with ferritin-conjugated lectin.