Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study.

A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.

Subclavian deep vein thrombosis associated with the use of recombinant follicle-stimulating hormone (Gonal-F) complicating mild ovarian hyperstimulation syndrome.

OBJECTIVE: To describe two cases of subclavian deep vein thrombosis (DVT) associated with the use of recombinant gonadotropins and mild ovarian hyperstimulation syndrome (OHSS) and review the literature associated with this condition. DESIGN: Retrospective study (case report). SETTING: Tertiary academic IVF program. PATIENT(S): Two women undergoing IVF with intracytoplasmic sperm injection due to male factor infertility. INTERVENTION(S): Ovaluation induction with recombinant FSH, IVF, and therapeutic heparinization. MAIN OUTCOME MEASURE(S): Coagulation studies, resolution of DVT, delivery at term. RESULT(S): Mild OHSS with left subclavian thrombosis occurred in two patients. Laboratory evaluation revealed normal protein C, protein S, antinuclear antibodies (ANA), and absence of antiphospholipid (APA) and anticardiolipin antibodies (ACA). Antithrombin III levels and coagulation studies were also within normal limits. Both patients tested negative for a factor V Leiden mutation and delivered healthy infants at term. CONCLUSION(S): Arm swelling associated with the use of gonadotropins during controlled ovarian hyperstimulation should be promptly evaluated and treated. Subclavian vein thrombosis is a rare complication of ovulation induction, and the possibility that recombinant gonadotropins increase the risk for this complication should be further studied.

Fifteen years experience with an in-vitro fertilization surrogate gestational pregnancy programme.

The purpose of our study was to review and evaluate retrospectively the experience of an in-vitro fertilization (IVF) surrogate gestational programme in a tertiary care and academic centre. In a 15 year period from 1984 to 1999, a total of 180 cycles of IVF surrogate gestational pregnancy was started in 112 couples. On average, the women were 34.4 +/- 4.4 years of age, had 11.1 +/- 0.72 oocytes obtained per retrieval, 7.1 +/- 0.5 oocytes fertilized and 5. 8 +/- 0.4 embryos subsequently cleaved. Sixteen cycles (8.9%) were cancelled due to poor stimulation. Except for six cycles (3.3%) where there were no embryos available, an average of 3.2 +/- 0.1 embryos was transferred to each individual recipient. The overall pregnancy rate per cycle after IVF surrogacy was 24% (38 of 158), with a clinical pregnancy rate of 19% (30 of 158), and a live birth rate of 15.8% (25 of 158). When compared to patients who underwent a hysterectomy, individuals with congenital absence of the uterus had significantly more oocytes retrieved (P < 0.006), fertilized, cleaved and more embryos available for transfer despite being of comparable age. IVF surrogate gestation is an established, yet still controversial, approach to the care of infertile couples. Take-home baby rates are comparable to conventional IVF over the same 15 year span in our programme. Patients with congenital absence of the uterus responded to ovulation induction better than patients who underwent a hysterectomy, perhaps due in part to ovarian compromise from previous surgical procedures.

Gonadotropins induce higher active renin levels in the follicular fluid of normal and hyperstimulated cycles.

The objective of this study was to analyze follicular fluid active renin and its relationship to steroid hormones throughout the normal and gonadotropin-stimulated menstrual cycle. Active renin was measured in the follicular fluid of patients undergoing tubal sterilization (n = 16) and in vitro fertilization (IVF) (n = 25); IVF patients were either in a natural cycle (n = 7) or undergoing controlled ovarian hyperstimulation (n = 18). The largest visible follicle was aspirated at the time of laparoscopic tubal sterilization; ultrasound guided transvaginal follicular aspiration was used in the IVF group. Follicular fluid active renin, estradiol and progesterone levels were measured with immunoradiometric and fluoroimmunoassays. The cycle day was correlated with the spontaneous luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) administration, as well as active renin, estradiol, progesterone levels and the estradiol/progesterone ratio using simple and multiple regression and analysis of variance (ANOVA). Cycle day independently influenced active renin, progesterone and the estradiol/progesterone ratio in a statistically significant manner (p < 0.0001). The active renin and progesterone levels were highest during the periovulatory period (p < 0.0001 and p < 0.002, respectively) and the estradiol/progesterone ratio correlated inversely with cycle day (p < 0.003). Although the follicular fluid active renin, estradiol and progesterone levels were higher after controlled ovarian hyperstimulation when compared to natural cycles, this difference did not reach statistical significance. Our findings suggest that active renin levels in follicular fluid increase in the follicular phase of the menstrual cycle, reaching peak levels in the periovulatory period following the LH surge or hCG administration, providing indirect support for the hypothesis that the ovarian renin-angiotensin system (RAS) is under gonadotropin control.

Gonadotropins induce the release of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha from the human preovulatory follicle.

PROBLEM: The effects of exogenous gonadotropin administration and steroid levels on the release of various cytokines into the human follicular fluid (FF) were studied. METHOD OF STUDY: Forty patients were included in two groups, those undergoing controlled ovarian hyperstimulation (COH) (n = 33) and natural cycles (n = 7). FF transvaginal aspirations were performed 36 hr after administration of human chorionic gonadotropin or a spontaneous surge of luteinizing hormone, respectively. FF cytokine measurements were performed with sensitive immunoassays. RESULTS: FF cytokine levels were higher after COH [interleukin (IL)-1 beta, 6.6 +/- 0.32 pg/ml; IL-6, 18.7 +/- 2.1 pg/ml; and tumor necrosis factor (TNF)-alpha, 32.5 +/- 4.9 pg/ml] than in natural unstimulated cycles (0.52 +/- 0.1 pg/ml, P < 0.001; 8.9 +/- 1.2 pg/ml, P < 0.01; and 13.2 +/- 2.6 pg/ml, P < 0.001, respectively). FF estradiol (E2) and progesterone levels were not statistically different between groups, despite the higher serum E2 levels observed in patients after COH. CONCLUSIONS: Gonadotropins might regulate ovarian secretion of cytokines, because FF IL-1 beta, IL-6, and TNF-alpha levels after COH were higher than during natural cycles.

Evaluation of predictive factors for multiple pregnancies during gonadotropin/IUI treatment.

PURPOSE: Our purpose was to analyze factors for their predictability of multiple pregnancies in patients treated with a combination of human menopausal gonadotropins (hMG) and intrauterine insemination (IUI). METHODS: The records of all patients conceiving through treatment with hMG/IUI were reviewed for factors predictive of multiple pregnancy. RESULTS: Ninety-one pregnancies reviewed included 78 singleton (86%), 8 twin (9%), and 5 higher-order pregnancies (5%). The total number of follicles > 10 mm was greater in the multiple-pregnancy group and there was a decreased number of postwash sperm in the multiple-pregnancy group. Otherwise there was no significant difference in the factors analyzed. CONCLUSIONS: No factor or combination of factors predicts multiple pregnancies to a degree that it would be helpful in managing or counseling patients.

Cost analysis of shared oocyte in vitro fertilization.

OBJECTIVE: To estimate the cost per woman delivered of a shared oocyte in an in vitro fertilization (IVF) program, and to compare these costs with those of a routine IVF program. METHODS: The cost of 23 shared oocyte cycles was analyzed retrospectively and compared with the cost of routine IVF. RESULTS: The cost per woman delivered in the shared oocyte program was approximately $22,000, compared with $66,000 in the routine IVF program. CONCLUSION: Shared IVF is a very cost-effective program and others donors and recipients an otherwise unavailable opportunity.

Limiting multiple pregnancy in in vitro fertilization/embryo transfer (IVF-ET) cycles.

PURPOSE: Our purpose was to assess how the number of embryos transferred can be adjusted to limit multiple gestations. METHODS: A retrospective analysis of 535 consecutive embryo transfers for the years 1991-1993 was conducted. RESULTS: Fewer than three embryos were associated with a low pregnancy rate. Pregnancy rates were highest in women less than 35 when four or more embryos were transferred. With four or more embryos, multiple gestation pregnancy correlated with the number of high-quality embryos transferred. The risk of triplets and quadruplets was greatest for women less than 40. CONCLUSIONS: Multiple-embryo transfer carries a risk of plural gestation. The risk of multiple pregnancy cannot be eliminated without decreasing the pregnancy rate. The risk of high-order multiple pregnancy was best correlated with the number of good-quality embryos transferred. While all are at risk, patients younger than 40 were at highest risk.

Growth factor/cytokine secretion by a permanent human endometrial cell line with embryotrophic properties.

OBJECTIVE: Our goal was to identify and quantitate growth factors and cytokines actively secreted by a permanent human endometrial cell line with embryotrophic properties and to determine if cell lines enhancing embryo development secrete common growth factors and/or cytokines. DESIGN: Culture medium conditioned by this human endometrial cell line was screened for platelet-derived growth factor (PDGF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) by enzyme-linked immunoassay (ELISA). Comparisons were made to the African Green monkey kidney epithelial cell line (Vero), which has been well documented to be embryotrophic. SETTING: This study was conducted in a university-based research laboratory associated with a clinical IVF program. RESULTS: The monkey Vero cell line and our permanent human endometrial cell line produced several common growth factors and cytokines, such as IL-6, PDGF, and LIF. IL-6 was secreted in substantial amounts by both Vero cells (400 pg/ml per 100,000 cells) and endometrial cells (100 pg/ml). PDGF levels in culture supernatant were measured to be 72 and 35 pg/ml, respectively, for Vero and endometrial cells. LIF could not be assayed in our human cell line by ELISA but its presence was evidenced by immunocytochemistry. EGF and active TGF-beta were not detectable in conditioned medium from either cell line. CONCLUSIONS: The two embryotrophic cell lines analyzed in this investigation synthesized several common growth factors and cytokines. Identification of factors being secreted by cell lines which positively influence embryo development may aid us in understanding the basic growth requirements of the preimplantation embryo.

Ovarian hyperstimulation syndrome: pre-ovulatory serum concentrations of interleukin-6, interleukin-1 receptor antagonist and tumour necrosis factor-alpha cannot predict its occurrence.

The pathogenesis of the ovarian hyperstimulation syndrome (OHSS) is poorly understood. Since significant elevations in cytokines are found in OHSS, our objective was to conduct a prospective case-controlled study to assess if pre-ovulatory cytokine serum concentrations can predict its occurrence. The study group was selected from in-vitro fertilization patients who subsequently developed severe OHSS, along with a matched group who did not develop this complication (n = 20), and a healthy normal control group (n = 10). Interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1RA) and tumour necrosis factor-alpha (TNFalpha) measurements were performed with sensitive immunoassays and confirmed with bioassays. Serum IL-6 (mean concentrations +/- SEM: 4. 38 +/- 0.36 pg/ml), IL-1RA (829 +/- 292 pg/ml) and TNFalpha (15.5 +/- 1.32 pg/ml) concentrations did not show differences throughout the normal menstrual cycle group. Cytokine variability and pre-ovulatory values were similar in OHSS compared to controlled ovarian hyperstimulation (COH) patients. However, average follicular phase serum IL-6 concentrations were higher in OHSS (8.71 +/- 0.41 pg/ml) and COH (7.66 +/- 0.38 pg/ml) patients than in normally menstruating women (4.34 +/- 0.99 pg/ml) (P < 0.0001). Pre-ovulatory serum IL-6 concentrations were also higher in OHSS (9 +/- 0.94 pg/ml) and COH (7.3 +/- 0.97 pg/ml) patients than in controls (4.57 +/- 1.1 pg/ml) (P < 0.01 and P < 0.04 respectively). All IL-1RA and TNFalpha concentrations were comparable in all the groups. This study suggests that cytokine measurements cannot be used to predict the occurrence of OHSS prior to the administration of human chorionic gonadotrophin.

Elevated interleukin-6 levels in the ovarian hyperstimulation syndrome: ovarian immunohistochemical localization of interleukin-6 signal.

OBJECTIVE: To examine the production and immunolocalization of interleukin-6 (IL-6) in patients with the ovarian hyperstimulation syndrome. METHODS: The study group consisted of patients with ovarian hyperstimulation syndrome (n = 9) from whom serum and ascites samples were obtained. The control samples used were serum (n = 10) and peritoneal (n = 16) and follicular fluids (n = 8) from healthy individuals. Follicular fluid (n = 40) and serial serum samples were also obtained from patients undergoing menotropin stimulation for in vitro fertilization (IVF) before (n = 10) and after ovulation (n = 34). Interleukin-6 measurements were performed with a sensitive immunoassay and confirmed with a bioassay. Immunohistochemical localization of IL-6 was performed with a mouse monoclonal antibody in normal premenopausal (n = 5) and postmenopausal ovaries (n = 5), as well as with cells from stimulated follicular fluid aspirates (n = 3). RESULTS: We found significantly higher serum and ascites IL-6 levels in ovarian hyperstimulation syndrome (mean 18.8 +/- 1.1 and 810.8 +/- 60.7 pg/mL, respectively) compared with postovulatory serum and peritoneal fluid from normal controls (mean 4.4 +/- .69 and 44.7 +/- 7.5 pg/mL, respectively) (P < .001) or serum after menotropin stimulation (13.1 +/- 1.1 pg/mL) (P < .001). At the time of ovulation, follicular fluid IL-6 levels (normal controls, mean 9 +/- 2.1 pg/mL; menotropin stimulation, mean 10.1 +/- 4 pg/mL) were higher than in preovulatory serum (normal controls, mean 4.5 +/- .8 pg/mL; menotropin stimulation, mean 6.3 +/- 1.4 pg/mL) (P < .001). Immunohistochemical localization of IL-6 revealed intense staining in corpora lutea and theca cells from large antral follicles and luteinized granulosa cells in follicular aspirates after menotropin stimulation. CONCLUSION: Interleukin-6 levels are markedly elevated in the ovarian hyperstimulation syndrome when compared with controls. The higher follicular fluid IL-6 levels seen suggest local secretion of this cytokine. Immunohistochemical correlation demonstrated IL-6 within ovarian theca cells. These findings suggest a local role for IL-6 both in normal and stimulated ovarian function. Whether IL-6 is directly responsible for the clinical manifestations of this syndrome is unclear. However, when produced in massive amounts, the pro-inflammatory effects of IL-6 may contribute to its pathogenesis and perhaps serve as a marker for the disease.

Cost-effectiveness of in vitro fertilization.

OBJECTIVE: To estimate the cost per woman delivered of pregnancies from an in vitro fertilization (IVF) program, and to assess what factors contribute to the cost. METHODS: The cost during 2 years' experience of a single IVF program was analyzed retrospectively. Cost analysis included cost of the IVF procedure itself as well as costs incurred because of maternal and neonatal complications. RESULTS: The major contributor to the total cost of IVF was high-order multiple pregnancies. The cost per woman delivered of singleton or twin pregnancies was approximately $39,000, whereas the cost per woman delivered of triplet and quadruplet pregnancies was approximately $340,000. CONCLUSION: In vitro fertilization can be cost-effective if steps are taken to minimize high-order multiple (triplet or more) pregnancies.

Clinical experience with synthetic serum substitute as a protein supplement in IVF culture media: a retrospective study.

OBJECTIVE: Our objective was to evaluate the efficacy of Synthetic Serum Substitute (Irvine Scientific--Materials Section, Santa Ana, CA), a globulin-enriched protein preparation containing human serum albumin for supplementation of IVF culture media. DESIGN: We retrospectively analyzed IVF cycles performed at MacDonald Womens Hospital between January 1992 and November 1994. IVF cycles were reviewed and classified according to the nature of protein supplementation used in the embryo culture medium. Three protein supplements utilized during this time period were compared: Synthetic Serum Substitute (SSS), Plasmanate (PL), and maternal serum (MS). RESULTS: Although clinical pregnancy rates among the three treatment groups were not statistically different, there was a definite trend toward a higher pregnancy rate with SSS supplementation (SSS, 38.2%; MS, 28.0%; and PL, 24.9%). Embryos grown in SSS-supplemented culture media had a significantly higher implantation rate (17.8 vs 10.4 and 10.3%, respectively, for MS and PL). Preliminary data also suggest that human embryo development and blastulation in vitro were enhanced by this protein supplement. CONCLUSIONS: The higher implantation rate with SSS suggests that it may be superior to both maternal serum and Plasmanate in supporting human embryo development in vitro. Whether blastocysts derived from PL- and SSS-supplemented media are able to implant and give rise to clinical pregnancies remains to be seen.

Cornual heterotopic pregnancy and cornual resection after in vitro fertilization/embryo transfer. A report of two cases.

BACKGROUND: Heterotopic pregnancy occurs in about 1% of pregnancies achieved with assisted reproductive technologies. The incidence of cornual pregnancy is approximately 1% of all ectopics. CASES: Two patients became pregnant after treatment with IVF-ET. Both presented during the first trimester with evidence of an acute abdomen and ultrasonographic suspicion for cornual heterotopic pregnancies. The diagnoses were confirmed by laparoscopy, and treatment was undertaken with laparotomy with cornual resection. Both delivered viable infants by cesarean section; the first patient delivered a twin gestation at 28 weeks and the second a singleton pregnancy at 37 weeks. All infants were doing well two months after delivery. CONCLUSION: Abnormal pregnancies can be detected after IVF-ET with careful follow-up, transvaginal ultrasound studies, serial hCG measurements and pelvic examinations. Cornual heterotopic pregnancies can be successfully treated with cornual resection, especially in cases presenting during the second trimester or when there is clinical evidence of an acute abdomen. All deliveries should be performed by cesarean section at term prior to labor or when tocolysis for premature labor has failed.

Elevated levels of interleukin-6 in ascites and serum from women with ovarian hyperstimulation syndrome.

OBJECTIVE: To examine the role of pro-inflammatory cytokines in ovarian hyperstimulation syndrome (OHSS). DESIGN: In vitro laboratory study of serum, peritoneal cells isolated and fluid obtained from ascites removed in the therapeutic management of four patients with OHSS. SETTING: Tertiary care referral teaching hospital. PATIENTS: Four patients with OHSS comprised the study population. Five healthy women at the time of elective laparoscopic tubal ligation served as controls. Control serum was also obtained from healthy adult volunteers, and control peritoneal fluid (PF) was obtained from patients on peritoneal dialysis. INTERVENTIONS: Therapeutic paracentesis was performed on four patients with OHSS. RESULTS: Peritoneal cells were isolated and fluid obtained from ascites removed in the therapeutic management of the women with OHSS. Peritoneal cells were obtained by intraoperative lavage from the control women. The cells were incubated with various concentrations of Escherichia coli lipopolysaccharide (LPS) for 20 and 48 hours. Interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were assayed in the incubation supernatants. The release of the three cytokines and PGE2 in response to LPS by peritoneal cells from women with OHSS was not different from the controls. However, both serum and ascitic fluid from women with OHSS contained significantly greater levels of IL-6 than control serum and PF. No significant differences in TNF levels in serum, ascitic fluid, or PF could be found by ELISA or bioassay. CONCLUSIONS: Increased production of IL-6, released into the peritoneal cavity and the circulation, may mediate OHSS.

Preliminary experience with in vitro fertilization-surrogate gestational pregnancy.

This report documents the medical details and 3-year follow-up evaluation of the infertile and surrogate couples involved in the first successful in vitro fertilization gestational surrogate pregnancy and summarizes clinical experience and outcome of all patients treated to date. Results of the first 28 couples treated are presented. The pregnancy rate for 39 cycles reaching attempts at oocyte retrieval is 18%. The procedural aspects, ethical issues, legal issues, and subsequent program development are summarized. Recommendations are of a similar program. There are numerous potential pitfalls and traps for the unwary, but our experience has thus far been gratifyingly positive, and we endorse the further provision, observation, and documentation of this controversial approach to the care of the infertile couple.

In vitro fertilization (IVF)-surrogacy: application of IVF to women without functional uteri.

Women with absent or dysfunctional uteri consented to controlled ovarian stimulation, ovum aspiration, in vitro fertilization, and embryo culture. Cleaving preembryos were transferred to recipient (surrogate) women whose menstrual cycles were in approximate synchrony with the ovum donor. None of the embryo recipients received medication. Six cases are described, resulting in one spontaneous loss at 6 weeks, four full-term deliveries, and one ongoing pregnancy. HLA typing demonstrated all babies to be genotypic offspring of the gamete donors.