Are cytokines and growth factors responsible for the detrimental effects of hydrosalpingeal fluid on pregnancy rates after in vitro fertilization-embryo transfer?

OBJECTIVE: To characterize the secretion of cytokines and growth factors in hydrosalpingeal fluid. DESIGN: Retrospective analysis. SETTING: Hospital-based infertility practice. PATIENT(S): Ten infertile women who underwent laparoscopic aspiration of their hydrosalpingeal fluid before salpingectomy or neosalpingostomy. INTERVENTION(S): Samples were cryopreserved, then thawed and centrifuged to remove cellular debris. MAIN OUTCOME MEASURE(S): The supernatants were analyzed for the presence of human interferon-gamma, epidermal growth factor, transforming growth factor-beta2, and tumor necrosis factor-alpha by quantitative enzyme immunoassay kits. RESULT(S): Interferon-gamma and transforming growth factor-beta2 were not detected in any of the hydrosalpingeal fluid samples. Epidermal growth factor was present in 5 of 10 hydrosalpingeal fluid samples, with a mean (+/- SE) concentration of 26.7+/-11.4 pg/mL. Tumor necrosis factor-alpha was detected in 7 of 10 samples, with a mean (+/- SE) concentration of 6.2+/-3.6 pg/mL. Three of the 10 samples contained both tumor necrosis factor-alpha and epidermal growth factor. CONCLUSION(S): For the first time, we described the absence of interferon-gamma and transforming growth factor-beta2, and the presence of epidermal growth factor and tumor necrosis factor-alpha in human hydrosalpingeal fluid. Because the fundamental role of the human fallopian tube is secretory in nature, the alteration in substances secreted from the tubal epithelium that reflux into the uterine cavity may explain the deleterious effects that hydrosalpingeal fluid has on pregnancy rates after IVF-ET.

Expression of the insulin-like growth factor-1 gene and its receptor in preimplantation mouse embryos; is it a marker of embryo viability?

Recent studies have demonstrated the importance of insulin-like growth factors (IGF) in mouse preimplantation development. We examined IGF-1 and IGF-1 receptor (IGF-1R) gene expression in a single blastomere of an early mouse embryo and compared it with subsequent embryo development in culture. Fertilized eggs and 2-cell embryos were obtained by tubal flushing in superovulated and mated female mice. Single cells were removed from embryos at cleavage stage between 3 and 8 cells using the standard embryo biopsy techniques. Individual blastomeres from each embryo were then assayed for the presence of IGF-1 and IGF-1R mRNA using reverse transcription-polymerase chain reaction. The biopsied embryos were washed in medium and placed in co-culture with murine endometrial cells. Embryonic development in culture was assessed and blastocyst grading was performed. IGF-1 gene expression was then examined for an association with in-vitro development. Eighty-seven embryos were biopsied. IGF-1R gene expression was detected in the majority of embryos tested and IGF-1 gene expression was detected in 34 of 81 (42%) embryos. A significant association between IGF-1 expression and blastocyst formation in vitro was found (P < 0.01). There was no association between IGF-1R expression and subsequent embryo development. We conclude that IGF-1 gene expression could potentially be used as a marker of embryo quality.

The effect of hydrosalpinges on IVF-ET outcome.

PURPOSE: Our purpose was to determine if the presence of a hydrosalpinx effects the outcome of in vitro fertilization (IVF)-embryo transfer. METHODS: We performed a retrospective analysis of IVF cycle stimulation sheets. RESULTS: A total of 1000 patients with tubal factor infertility was analyzed. There were 60 hydrosalpinx patients who underwent 116 initiated cycles with 106 embryo transfers, compared to 940 control patients undergoing 1428 initiated cycles with 1150 embryo transfers. Both groups had a similar response to ovarian stimulation, number of oocytes retrieved, and number of embryos transferred. The hydrosalpinx group had a significantly higher preclinical loss rate (22/59 = 37% vs 80/566 = 14%; P = 0.001), a significantly lower implantation rate (55/352 = 16% vs 795/3795 = 21%; P = 0.013), a trend toward a reduced delivery rate per transfer (28/106 = 26% vs 387/1150 = 34%; P = 0.066), a significantly higher ectopic pregnancy rate (5/59 = 8% vs 16/566 = 3%; P = 0.04), and a similar spontaneous abortion rate (9/37 = 24% vs 99/486 = 20%; P = 0.28) compared to the control tubal factor group. CONCLUSIONS: This study demonstrates a decrease in implantation rates and an increase in preclinical miscarriages and ectopic pregnancies in patients with hydrosalpinges compared to tubal-factor patients without sonographic evidence of dilated fallopian tubes.

The embryo toxicity of hydrosalpinx fluid is only apparent at high concentrations: an in vitro model that stimulates in vivo events.

OBJECTIVE: To simulate the in vivo model in studying the effect of hydrosalpinx fluid on embryonic development. DESIGN: Controlled prospective study. SETTING: Academic research center. PATIENT(S): Five hundred eighty-seven two-cell murine embryos. INTERVENTION(S): Embryos were grown under two sets of conditions. Half were cultured using 10% fetal calf serum in RPM1 medium in varying concentrations of hydrosalpinx fluid (0, 1%, 10%, 50%, 75%, and 100%). To more closely mimic the in vivo environment, the other half were grown in an endometrial coculture system with the same media and hydrosalpinx fluid concentrations. MAIN OUTCOME MEASURE(S): Embryonic development. RESULT(S): For each stage of embryogenesis, diminished development was noted with increasing concentrations of hydrosalpinx fluid. In the group of embryos grown without endometrial coculture, only at a minimum concentration of 50% hydrosalpinx fluid was diminished development noted for the blastocyst, hatching, and outgrowth stages. When an endometrial coculture system was used, development was not inhibited until exposure to a minimum of 75% hydrosalpinx fluid. Embryogenesis was enhanced when an endometrial coculture system was used for each concentration of hydrosalpinx fluid. CONCLUSION(S): When a model is used that more accurately mimics the in vivo conditions of IVF-ET in a patient with hydrosalpinges, it appears that high concentrations of hydrosalpinx fluid are required to signiticantly impede embryogenesis. The endometrium appears to help detoxify hydrosalpinx fluid.

Expression of inhibin/activin subunits and their receptors and binding proteins in human preimplantation embryos.

PURPOSE: Our purpose was to study the role of inhibin/activin during embryogenesis. METHODS: Transcripts of inhibin/activin subunits (alpha, beta A, beta B), activin receptors (types I and II), and follistatin were detected by a reverse transcriptase-polymerase chain reaction in human reproductive cells and preembryos cultured alone or co-cultured with human endometrial cells. RESULTS: Transcripts of alpha, beta A, beta B subunits were all detected in granulosa luteal cells, but only beta A units were detected in endometrial stromal and decidualized cells. In human preimplantation embryos, none of these subunits were detected in embryos from the four-cell to the morula stage and only beta A subunits were detectable in blastocyst embryos. Activin receptors were detectable in all of the studied embryos and cells. Transcripts of beta A, activin receptors, and follistatin were differentially expressed in human preimplantation embryos cultured in vitro and their expressions were significantly enhanced with the presence of endometrial stromal cells. CONCLUSIONS: Our data suggest that there is a possible endometrium-embryo interaction via endometrial activins and preimplantation embryo receptors and that the embryonic expressions of these activins, their receptors, and binding proteins are dependent on embryonic stage.

Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer.

PURPOSE: Our purpose was to evaluate the effect of coculture on preembryo development and clinical outcome. METHODS: Enrolled patients underwent a luteal-phase endometrial biopsy. The tissue was then enzymatically digested (collagenase) and the stromal and glandular cells were separated by differential sedimentation rates. These cells were cultured to confluence, released, and then cryopreserved until the patient's in vitro fertilization (IVF)-embryo transfer (ET) cycle. All normally fertilized oocytes were then placed on the co-cultured cells until transfer on day 3. Preembryo development on co-culture was compared to that in the patient's noncocultured previous cycle. Implantation and clinical pregnancy rates were compared to those in a control group of patients undergoing IVF during the study period who were matched for age, stimulation protocol, number of oocytes retrieved, and preembryos transferred. RESULTS: Twenty-nine women underwent 31 cycles of IVF-ET. On day 3 the overall mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.3 +/- 1.8 vs. 5.6 +/- 1.2 (P = 0.04). The average percentage of cytoplasmic fragments on co-culture compared to the previous cycle was 16 +/- 9% vs. 19 +/- 9% (P = 0.32). At transfer, after preembryo selection, the mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.8 +/- 1.6 vs. 6.6 +/- 1.3 (P = 0.5). The implantation and clinical pregnancy rates between co-culture and the matched control group were 15% (14/93) vs. 13% (16/124) (P = 0.79) and 29% (9/31) vs. 25% (10/40) (P = 0.45). CONCLUSIONS: There was a significant improvement in the average number of blastomeres per preembryo on co-culture compared to that in the patient's previous noncoculture cycle. The overall implantation and clinical pregnancy rates between co-culture and a matched control group were not significantly different.

Granulocyte macrophage-colony stimulating factor production by autologous endometrial co-culture is associated with outcome for in vitro fertilization patients with a history of multiple implantation failures.

PROBLEM: To determine whether granulocyte macrophage (GM)-colony stimulating factor (CSF) produced by autologous endometrial co-culture was associated with outcome in 53 patients with a history of multiple in vitro fertilization failures. METHOD OF STUDY: The conditioned media from endometrial co-culture cells exposed or non-exposed to human embryos was analyzed for GM-CSF. RESULTS: Exposure or non-exposure to an embryo did not result in an enhancement of GM-CSF production. Insignificant levels of GM-CSF were determined from media alone. ROC analysis revealed that levels of GM-CSF from supernatants of endometrial co-culture exposed to embryos that measured below 130 pg/ml reflected a diminished prognosis (5/17 had a positive pregnancy vs. 21/36 with GM-CSF levels greater than 130 pg/ml; P < 0.05). CONCLUSIONS: The improved outcome associated with GM-CSF values greater than 130 pg/ml may reflect: 1) a direct positive effect of GM-CSF; 2) an embryotrophic factor upregulated by GM-CSF; or, 3) that GM-CSF functions as a marker for the importance of the glandular component in endometrial co-culture systems.

Human preembryo development on autologous endometrial coculture versus conventional medium.

OBJECTIVE: To evaluate the effect of autologous endometrial coculture versus conventional medium on preembryo development. DESIGN: Controlled systematic clinical study. SETTING: University-based IVF center. PATIENT(S): Women with a history of failed IVF-ET with poor preembryo quality. INTERVENTION(S): Patients underwent a luteal phase endometrial biopsy. The tissue then was digested enzymatically, and the stromal and glandular cells were separated by differential sedimentation rates. These cells were cultured to confluence, released, and then cryopreserved until the patient's IVF-ET cycle. All normally fertilized oocytes then were allocated systematically to growth on autologous endometrial coculture or conventional medium until transfer on day 3. MAIN OUTCOME MEASURE(S): Preembryo blastomere numbers and cytoplasmic fragmentation rates were measured. RESULT(S): Forty-two women underwent 44 cycles of IVF-ET. In the morning on day 3, the mean (+/-SD) number of blastomeres and cytoplasmic fragments per preembryo on coculture compared with conventional medium was 5.9+/-1.5 versus 5.5+/-1.4 and 21%+/-13% versus 24%+/-11. At transfer the mean (+/-SD) number of blastomeres per preembryo on coculture was 7.4+/-1.3 versus 6.7+/-1.9 on conventional medium. CONCLUSION(S): There was a significant improvement in the mean (+/-SD) number of blastomeres per preembryo and decrease in the fragmentation rate for preembryos on autologous endometrial coculture compared with noncocultured preembryos from the same patient.

Comparison of clinical outcome after cryopreservation of embryos obtained from intracytoplasmic sperm injection and in-vitro fertilization.

The impact of intracytoplasmic sperm injection (ICSI) on cryopreserved zygotes and embryos was evaluated by comparing embryo survival and implantation between embryos derived from ICSI and those derived from standard insemination procedures. The study included patients whose excess zygotes and embryos were cryopreserved between September 1993 and December 1994 and who subsequently underwent a frozen embryo transfer. Embryo survival, clinical pregnancy rates per transfer and pregnancy outcome were compared. Three hundred and thirty eight cryopreservation cycles, during which 1471 embryos were cryopreserved, were included in this study. Of those, 961 were derived from oocytes fertilized by insemination in vitro and 510 were derived from oocytes fertilized by ICSI. A total of 690 of the embryos (451 in the insemination group and 239 in the ICSI group) have since undergone a thaw cycle. The embryo survival rates were similar between the two groups (70.5 and 73.2%, insemination and ICSI respectively) and were not significantly affected by the stage at cryopreservation. There was no significant difference in pregnancy rates per transfer (31.8 and 32.3%), the preclinical pregnancy loss rate (16.7 and 23.8%), or the clinical miscarriage rate (16.7 and 23.8%) between the insemination and the ICSI groups respectively. It is concluded that ICSI does not have an adverse impact on the survival and successful implantation of cryopreserved and thawed embryos.

Oocyte donation: does a previous attempt affect a subsequent attempt?

OBJECTIVE: To analyze the effect of a previous donor oocyte cycle on the outcome of subsequent attempts. DESIGN: Retrospective study. SETTING: Oocyte donation program at The New York Hospital/Cornell Medical Center. PATIENT(S): Two hundred sixty-seven patients undergoing 354 fresh cycles of oocyte donation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical outcomes were divided into groups based on the attempt number of each cycle for each patient. Results were calculated for each recipient cycle. RESULT(S): A clinical pregnancy rate of 56.2% and ongoing pregnancy/delivery rate per retrieval of 50.3% were noted. No statistically significant differences in clinical outcomes were found between the first, second, and third attempts. A significant increase was noted in the ongoing pregnancy/delivery rate per recipient cycle for the second attempt in those patients who had a delivery after the first attempt compared with those who did not. CONCLUSION(S): We demonstrated an overall clinical pregnancy rate of 56.2% and an ongoing pregnancy/delivery rate of 50.3% per retrieval. Outcome for the second attempt was associated with success or failure during an initial attempt at oocyte donation.

Ovarian estradiol production in vivo. Inhibitory effect of leuprolide acetate.

OBJECTIVE: To determine the impact of reducing the dose of gonadotropin-releasing hormone agonist (GnRH-a) for controlled ovarian stimulation in in vitro fertilization (IVF) on subsequent response to stimulation and cycle outcome. STUDY DESIGN: An IVF database was searched to identify patients who underwent at least two cycles of ovarian stimulation at a university-based medical center, and a retrospective chart review was performed. Fifty-one patients whose IVF stimulation protocols during the two cycles were identical except for the leuprolide dosage utilized for luteal pituitary suppression were included in the study. Two leuprolide dosages were utilized for suppression: a low dose, 0.5 mg daily, and a high dose, 1 mg daily. The leuprolide dose was uniformly halved upon initiation of gonadotropin stimulation; the gonadotropin dose and preparation were identical in the two protocols. Day 3 follicle-stimulating hormone levels, duration of stimulation, amount of gonadotropins required, midcycle and peak estradiol levels, oocyte yield and implantation rates were compared. RESULTS: Lowering the dose of GnRH-a while maintaining the same stimulation protocol resulted in a faster estradiol rise and higher mean peak estradiol level. The higher estradiol levels were obtained with a lower total gonadotropin dose. The oocyte yield was not affected by the stimulation protocol utilized. CONCLUSION: Lowering the dosage of leuprolide allows higher estradiol levels in those patients. This suggests an inhibitory in vivo effect of leuprolide acetate on ovarian steroidogenesis.

Growth factor expression by human oviduct and buffalo rat liver coculture cells.

OBJECTIVE: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth. DESIGN: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors. SETTING: Academic tertiary care hospital. PATIENT(S): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts. INTERVENTION(S): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5' end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography. RESULT(S): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF. CONCLUSION(S): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development.

Dual suppression with oral contraceptives and gonadotrophin releasing-hormone agonists improves in-vitro fertilization outcome in high responder patients.

Certain patients have a tendency for high response to gonadotrophin therapy which is often not ameliorated with prior gonadotrophin-releasing hormone agonist (GnRHa) suppression. As a result, these patients are frequently cancelled and often experience ovarian hyperstimulation syndrome (OHSS) episodes during in-vitro fertilization (IVF)-embryo transfer cycles. Patients with polycystic ovarian syndrome (PCOS) have been noted to be particularly sensitive to exogenous gonadotrophin therapy. We have developed a protocol which is effective in improving IVF outcome in high responder patients, including those with PCOS. Oral contraceptive pills (OCP) are taken for 25 days followed by s.c. leuprolide acetate, 1 mg/day, which is overlapped with the final 5 days of oral contraceptive administration. Low-dose gonadotrophin stimulation is then initiated on the third day of withdrawal bleeding in the form of either human menopausal gonadotrophins or purified urinary follicle-stimulating hormone at a dosage of 150 IU/day. Over a 5 year period, we reviewed our experience utilizing this dual method of suppression in 99 cycles obtained in 73 high responder patients. There were only 13 cancellations prior to embryo transfer (13.1%). The clinical and ongoing pregnancy rates per initiated cycle were 46.5 and 40.4% respectively. Only eight patients experienced mild-moderate OHSS following treatment. For those patients who had undergone previous IVF-embryo transfer cycles at our centre, significant improvements were noted in oocyte fertilization rates, embryo implantation rates and clinical/ongoing pregnancy rates with this protocol. Hormonal analyses revealed that the chief mechanism may be through an improved luteinizing hormone/follicle-stimulating hormone ratio following dual suppression. An additional feature of this dual method of suppression is significantly lower serum androgen concentrations, particularly dehydroepiandrosterone sulphate.

PIP: Presented is a protocol that is effective in improving in vitro fertilization (IVF) outcome in women with a tendency for a high response to gonadotrophin therapy. High responders to exogenous gonadotrophin therapy show recruitment of large numbers of follicles, rapid estradiol responses, and a significant cycle cancellation rate due to the potential risk of hyperstimulation during IVF-embryo transfer attempts. Women with polycystic ovarian syndrome are especially sensitive to exogenous gonadotrophin therapy. The protocol entails 25 days of oral contraceptive (OC) use, followed by 1 mg/day of subcutaneous leuprolide acetate overlapped with the final 5 days of OC therapy. On the third day of withdrawal bleeding, gonadotrophin stimulation is initiated through either human menopausal gonadotrophins or purified urinary follicle-stimulating hormone (150 IU/day). This approach permits normalization of the luteinizing hormone/follicle-stimulating hormone ratio and reduces ovarian androgen concentrations, while circumventing the initial gonadotrophin flare response. This protocol was tested in a retrospective review (1990-94) of 99 cycles from 73 high-responder women treated at a US infertility center. There were only 13 cancellations (13.1%) prior to embryo transfer. The clinical and ongoing pregnancy rates per initiated cycle were 46.5% and 40.4%, respectively. Only 8 women experienced ovarian hyperstimulation syndrome after treatment. Among women who had undergone previous IVF embryo transfer cycles at the center, the present regimen was associated with significant improvements in oocyte fertilization rates, embryo implantation rates, and pregnancy rates.

Twin gestation occupying separate horns of a bicornuate uterus after in-vitro fertilization and embryo transfer.

The detection of congenital uterine anomalies has increased because of heightened physician awareness and improved diagnostic modalities. The occurrence of a twin pregnancy occupying separate horns of a bicornuate uterus has been reported only sporadically in the literature. This is the first reported case resulting after in-vitro fertilization and embryo transfer.

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase.

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.