Successful pregnancy outcome after cryopreservation of all fresh embryos with subsequent transfer into an unstimulated cycle.

OBJECTIVE: To assess the pregnancy outcome of freezing and storing all fresh embryos produced in a stimulated IVF cycle and replacing them in a subsequent nongonadotropin-stimulated cycle. DESIGN: Retrospective study. SETTING: University-associated assisted reproductive technology program. PATIENTS: We studied 36 patients (age range 23 to 44 years) who underwent cryopreservation of all fresh embryos in a controlled ovarian hyperstimulation (COH) cycle because of either the risk of severe ovarian hyperstimulation (24 patients, group 1) or the presence of an endometrial lining < 8 mm in thickness (12 patients, group 2). Five hundred fifty-five embryos were generated for replacement in 63 cycles. All embryos were cryopreserved in 1.5 M propanediol at the pronuclear or two-cell stage, and 264 embryos subsequently were transferred into a hormone replacement cycle (70%) or natural ovulatory cycle (30%). The average number of embryos transferred per patient was 4.2. RESULTS: Twenty-one clinical pregnancies were achieved, giving a pregnancy rate (PR) of 58.3% per patient (33.3% per cycle). The live birth rate was 50% per patient (28.6% per cycle). The implantation rate was 9.1%. Groups 1 and 2 had a similar PR per patient (58.3%). With 208 cryopreserved embryos remaining and considering the 33.3% PR per cycle, we expect the overall extrapolated PR to be 63.9%. CONCLUSIONS: This is the first series showing that freezing and storing all fresh embryos produced in a stimulated IVF cycle and replacing them in a subsequent nongonadotropin-stimulated cycle results in successful PRs. These results underlie the importance of a successful cryopreservation program in IVF and could be a possible approach to overcoming the alleged adverse effects of COH on the endometrium, thereby improving the chances of pregnancy when numerous embryos are obtained simultaneously.

The use of progestins for programming assisted reproductive cycles and gonadotropin-releasing hormone agonist flare-up protocols in older patients.

OBJECTIVES: To determine if the use of norethisterone acetate (NET) in the previous cycle affects the ovarian response to GnRH agonist (GnRH-a) in flare-up protocols and controlled ovarian hyperstimulation in older patients. DESIGN: Retrospective analysis of the outcome of the assisted reproductive technology (ART) cycle. PATIENTS: Eighty women > 37 years old undergoing controlled ovarian hyperstimulation (COH) for a ART cycle (GIFT, IVF, zygote intrafallopian transfer). Forty received NET during the luteal phase of the previous cycle for programming the procedure and 40 did not receive NET (control group). Gonadotropin-releasing hormone agonist in follicular phase (flare-up) protocols were administered to all the patients. MAIN OUTCOME MEASURES: Cycle outcome: amount of gonadotropins used, oocyte production and quality, fertilization, and pregnancy rates. Estradiol, FSH, and LH levels the first 3 days of COH in eight patients. RESULTS: There were no differences between both groups in the cycle outcome. Estradiol levels during the first 3 days of COH were higher in the patients that did not receive NET in the previous cycle. Follicle-stimulating hormone and LH levels were similar in both groups. CONCLUSIONS: The administration of NET in the previous cycle in patients > 37 years old does not affect the ovarian response to the combination of follicular phase GnRH-a and gonadotropins for COH.

Outcome of triplet pregnancies after assisted reproductive techniques: how frequent are the vanishing embryos?

OBJECTIVE: To assess the incidence of spontaneous embryo reduction as well as the obstetric and neonatal outcome of triplet gestations after assisted reproductive techniques (ART). METHODS: We analyzed the spontaneous outcome of 38 pregnancies in which three gestational sacs were identified with vaginal ultrasound between 21 and 28 days after ART. Weekly follow-up visits were scheduled during the first trimester until referral to a high-risk obstetrician. After delivery, each patient was interviewed individually and, if necessary, the obstetrician was contacted. RESULTS: The triplets delivery rate was 47.4%, whereas 31.6% delivered twins, 18.4% delivered singletons, and only one patient miscarried all three cases (2.6%). Finding three fetal heart beats was associated with a triplet delivery rate of 69.2%, a twin incidence of 19.2%, and a singleton birth rate of 11.6%. Embryo resorptions were observed mainly during the first 7 weeks of gestation and did not occur beyond the 14th week. The mean gestational age at delivery and neonatal birth weight were significantly lower among triplets (32.8 weeks and 1,740 g versus 35.3 weeks and 2,352 g in twins and 39.1 weeks and 3,122 g for singletons). Triplets had a 100% prematurity and cesarean section rate compared with 67% and 75% in twins and 0% and 43% in singletons, respectively. Hospitalization at the Neonatal Intensive Care Unit was required in 83% of newborn triplets, 29% of twins, and 0% of singletons, with a mean stay of 34 and 21 days for triplets and twins, respectively. One stillbirth and no neonatal deaths were reported, with an overall perinatal mortality rate of 11.9 per 1,000. CONCLUSIONS: Spontaneously, approximately 50% of triplet pregnancies will experience at least one embryo resorption. The ongoing triplets demand a complex and more expensive perinatal management, a strong argument to consider limiting the number of oocytes-embryos transferred in ART.

Is there a role for ovarian stimulation and intra-uterine insemination after age 40?

The objective of this study was to determine the conception rate in infertile couples in which the female partner was > or = 40 years old and who had received ovarian stimulation treatment and intra-uterine insemination (IUI). It was a retrospective study of 77 patients who underwent a total of 210 treatment cycles. Protocols for ovulation induction included clomiphene citrate, human menopausal gonadotrophin (HMG) and clomiphene citrate plus HMG. Patients were monitored using transvaginal ultrasound, and two IUI were performed 24 and 48 h after the determination of urinary luteinizing hormone (LH) surge or human chorionic gonadotrophin (HCG) injection. A total of 11 pregnancies were reported, giving a pregnancy rate of 14% per patient and 5% per cycle. Eight spontaneous abortions occurred, giving a pregnancy wastage of 73%. In a previous comparative analysis of 543 patients < 39 years old receiving IUI and identical protocols of ovarian stimulation, 141 pregnancies were achieved, giving a pregnancy rate of 21% per patient and 10% per cycle. The miscarriage rate in that group was 18%. This report compares IUI results for women > or = 40 years with those obtained previously for younger women, and shows the very poor success rate in women > 40 years of age. This information will be important in the proper counselling of this group of patients, as well as indicating that a prompt recommendation for assisted reproductive treatment should be made soon after the failure of a few attempted cycles of ovarian stimulation treatment and IUI.

The predictive value of a single beta human chorionic gonadotropin in pregnancies achieved by assisted reproductive technology.

OBJECTIVE: To investigate whether a single serum beta-hCG in pregnancies achieved by assisted reproductive technologies (ART) can accurately predict pregnancy viability and, in viable pregnancies, multiple gestation. DESIGN: Four hundred sixty-one consecutive successful ART pregnancies were studied retrospectively. Seventy-one of the 461 patients were excluded because their beta-hCG was either drawn on the incorrect day or outside our facility. Three hundred ninety subjects had a serum beta-hCG drawn 14 days after ET or 16 days after gamete transfer. The beta-hCG samples were analyzed by immunoradiometric assay based on the Third International Reference Standard (IRS) (First International Reference Preparation (IRP)). Pregnancy status was followed, at minimum, through the first trimester. RESULTS: One hundred fifty (38%) of the 390 were found to be nonviable, resulting in spontaneous abortion (n = 38, 10%), ectopic pregnancy (n = 27, 6%), or biochemical pregnancies (n = 85, 22%). A statistically significant difference by the Scheffe F-test was found between the mean beta-hCG value of the nonviable (115 mIU/mL) (conversion factor to SI unit, 1.00) and viable (428 mIU/mL) pregnancies. The positive predictive value of a single beta-hCG > 100 mIU/mL in distinguishing viable from nonviable pregnancies was 0.83 (sensitivity 91%, specificity 71%). Of the 240 viable pregnancies, 74 (32%) were multiple gestations (57 twins, 14 triplets, and 3 quadruplets). The mean beta-hCG of the singleton pregnancies (266 mIU/mL) was significantly different from that of the multiple gestations (792 mIU/mL). The positive predictive value of a single serum beta-hCG < or = 400 mIU/mL in distinguishing singleton from multiple gestations was 0.92 (sensitivity 86%, specificity 82%). CONCLUSION: A single early serum beta-hCG may be used in ART pregnancies to predict which pregnancies will continue beyond the first trimester and to identify multiple gestations. Early reassuring tests may reduce anxiety.

Frozen zygote intrafallopian transfer: a successful approach for transfer of cryopreserved embryos.

OBJECTIVE: To assess whether frozen ET to the fallopian tube is a possible alternative for cryopreserved embryos. DESIGN: Fifty-four patients (mean age 35 years) participated, in which their embryos were cryopreserved in 1.5 M propanediol at the pronuclear or two-cell stage. Each patient then underwent a steroid replacement cycle consisting of oral micronized 17 beta-E2 2 mg on days 2 to 4, 4 mg on days 5 to 7, 6 mg on days 8 to 10, and 8 mg from day 11 on. Serial ultrasounds were performed to evaluate the endometrium until an optimal thickness of > or = 10 mm triple layer was achieved. At this time, 100 mg IM progesterone was initiated and the zygote intrafallopian transfer (ZIFT) procedure was performed on the third day of P administration. The average number of embryos transferred was 4.4. RESULTS: Twenty-two clinical pregnancies resulted, giving a pregnancy rate of 41%. Eight miscarriages occurred and one ectopic pregnancy resulted, giving a live birth rate of 24%. Implantation rate was 10.8%. The highest chance of pregnancy was seen in patients who never had a previous IVF, GIFT, or ZIFT (61%). CONCLUSION: This is the first report of a series of frozen ETs to the fallopian tubes. These results indicate that tubal transfer may offer a protective benefit of the tubal environment and avoidance of endometrial trauma, and should be added to our armamentarium of replacing cryopreserved embryos.

Prospective randomized comparison of human chorionic gonadotropin versus intramuscular progesterone for luteal-phase support in assisted reproduction.

OBJECTIVE: The purpose of the study was to determine the impact of two forms of luteal-phase supplementation, human chorionic gonadotropin (hCG) and progesterone (P), during gonadotropin releasing hormone agonist (GnRh-a)/controlled ovarian hyperstimulation (COH) cycles. DESIGN AND PATIENTS: The study was a prospective, randomized evaluation of 77 patients. Group 1 patients (n = 38) received 2000 IU of hCG, injected subcutaneously, on days 3, 6, 9, and 12 after transvaginal aspiration of the oocytes (TVA = day 0). Group 2 patients (n = 39) received 50-mg daily injections of intramuscular (i.m.) P from days 2 to 14 after TVA. Blood tests were performed on days 0, 5, 8, and 12 after TVA. SETTING: The in vitro fertilization program of a tertiary care institution was the study setting. MAIN OUTCOME MEASURES: The main outcome measures were (1) pregnancy and implantation rates; (2) serum estradiol (E), P, and hCG levels; and (3) occurrence of side effects. RESULTS: Clinical pregnancy and implantation rates in group 1 versus group 2 were similar (36.7 vs 35.3 and 12 vs 14%, respectively). Regardless of pregnancy occurrence, on days 8 and 12 after TVA, serum E and P levels were higher in group 1 than group 2 but the resulting E/P ratios were similar. Five of 38 patients (group 1) developed moderate to severe ovarian hyperstimulation syndrome (OHSS) right after the first or second supplementary hCG injection. In these patients, the mean serum E level on the day of hCG trigger injection was about 3250 pg/ml and the number of follicles was between 9 and 17. In 6 of 39 patients (group 2) allergic reactions were observed at the P injection sites. CONCLUSIONS: Based on our data, hCG administration as a form of luteal supplementation did not translate, in comparison to P, into significant benefits for the patients. At the same time, it significantly increased the risk of ovarian hyperstimulation. We suggest that whenever, during COH cycles, serum E levels are over 2500 pg/ml and the number of follicles exceeds 10, luteal support with hCG should be excluded.

The use of intravenous albumin in patients at high risk for severe ovarian hyperstimulation syndrome.

Previous experiences in subjects with other forms of third space fluid accumulation have shown that albumin is efficacious in preventing and correcting haemodynamic instability. Using a similar approach in an effort to increase the serum oncotic pressure and to reverse the leakage of fluids from the intravascular space, high risk subjects for severe ovarian hyperstimulation syndrome (SOHS) were treated with albumin. In a recent large study two high risk factors were identified, i.e. the number of oocytes and levels of serum oestradiol. Thirty-six women undergoing assisted reproductive techniques who presented both these factors, received intravenous albumin at a dose of 5% in Ringers lactate in doses of 500 ml during oocyte retrieval and 500 ml immediately thereafter in the recovery room. Daily measurements of urine output, serum and urine electrolytes, weight, abdominal girth, and haematocrit prior to and after oocyte retrieval revealed normal serum and urine electrolyte levels, and no signs of haemoconcentration. No patient in this study developed SOHS, and of course none had to be hospitalized. Vaginal ultrasound performed in the majority of the subjects revealed < or = 100 ml of peritoneal fluid 48-72 h after oocyte retrieval. The only complication from the use of intravenous albumin was the appearance of a 'flu-like condition' (low grade temperature, nausea and muscle pains) developed by 12 women between days 3 and 5 after oocyte collection. Intravenous albumin had thus prevented the development of severe ovarian hyperstimulation syndrome in an assisted reproduction programme.(ABSTRACT TRUNCATED AT 250 WORDS)

Routine use of vaginal ultrasonography in the preoperative evaluation of gynecologic patients. An adjunct to resident education.

To determine the clinical utility of routine vaginal ultrasound in evaluating gynecologic patients, we prospectively studied 133 women scheduled to undergo elective surgery based upon abnormal clinical findings as determined by residents and faculty in a large, county-based teaching hospital. Patients were examined bimanually by the surgeons and then examined ultrasonographically using a 5.0-MHz vaginal probe. Surgery was scheduled within 48 hours of the clinical examinations. Vaginosonography demonstrated enhanced sensitivity, specificity and predictive value as compared to bimanual pelvic examination. The technique was quickly mastered by resident physicians, with discrepancies between preoperative ultrasound observations and actual surgical findings present in but 15 of 532 instances. Vaginal ultrasonography is an effective routine adjunct to physical examination in the preoperative evaluation of surgical patients. Furthermore, it has diagnostic capabilities superior to those of the bimanual pelvic examination and increases the accuracy of preoperative diagnoses in gynecologic patients.

The effect of leuprolide acetate on steroidogenesis by granulosa and theca cells in vitro.

Enhancement of follicular response to controlled ovarian hyperstimulation for human in vitro fertilization (IVF) has been suggested following pretreatment with leuprolide acetate (LA). However, additional human menopausal gonadotropin (hMG) is required to achieve follicle maturity in the presence of LA. We studied the effect of LA on steroidogenesis of granulosa and theca cells in vitro. Human granulosa cells obtained from IVF follicular fluid aspirations were cultured for 14 days in the presence and absence of human chorionic gonadotropin (hCG). hCG significantly enhanced progesterone (P) and estradiol (E2) production by the cells, however, the addition of LA in concentrations of 10, 100, and 1000 ng/ml had no effect. Porcine granulosa cells were cultured for 48 hr in the presence and absence of follicle stimulating hormone (FSH) with the addition of LA at the same doses. LA did not affect the FSH-induced increase in P production. Porcine theca cells were cultured for 48 hr in the presence and absence of hCG. The addition of LA did not affect androstenedione (A) production by these cells. We conclude that in this dynamic model in vitro, LA does not inhibit or stimulate P or E2 production by granulosa or A production by theca cells.

Preovulatory follicular fluid steroid levels in stimulated and unstimulated cycles triggered with human chorionic gonadotropin.

The purpose of this study was to analyze follicular fluid (FF) samples for steroid levels from stimulated and unstimulated cycles triggered with human chorionic gonadotropin (hCG) and to assess the influence of controlled ovarian hyperstimulation and luteinizing hormone/hCG on these levels. Spontaneous ovulatory cycles were monitored with serial ultrasound examinations, and hCG 10,000 IU was given when the lead follicle was mature. Fourteen FF samples yielding fertilizable oocytes were compared with 13 FF samples from controlled ovarian hyperstimulation cycles. Progesterone (P) was higher in controlled ovarian hyperstimulation than in unstimulated cycles (9.0 +/- 1.2 micrograms/mL versus 4.4 +/- 0.6 microgram/mL; mean +/- SEM), whereas estradiol (E2) was lower (0.8 +/- 0.1 microgram/mL versus 1.3 +/- 0.2 microgram/mL), resulting in a higher P:E2 ratio (15.5 +/- 3.3 versus 4.4 +/- 0.7). Androstenedione (A), testosterone (T), and T:E2 ratios were all higher in unstimulated than controlled ovarian hyperstimulation cycles. We conclude that controlled ovarian hyperstimulation is associated with increased FF P, decreased FF E2, T, and A levels, and decreased T:E2 ratios, suggesting altered steroidogenesis and enhanced follicular aromatase activity.

Random urinary pregnanediol glucuronide measurements in pregnancy: lack of utility for evaluation of first-trimester vaginal bleeding.

Progesterone and its urinary metabolite pregnanediol-3 alpha-glucuronide (PDG) are generally lower in women with abnormal pregnancies compared to those with normal intrauterine gestations. We evaluated the ability of random urinary PDG measurements determined by enzyme immunoassay (EIA) to differentiate normal from abnormal pregnancies. Patients with first-trimester vaginal bleeding (n = 104) were evaluated. Eventual outcomes indicated 39 women had viable intrauterine pregnancies (IUPs), 54 had spontaneous abortions (SABs) and 11 had ectopic pregnancies (EPs). Urinary PDG was significantly lower in SAB and EP compared to IUP patients. However, a wide range of values in IUP patients was noted (3.2-93.3 micrograms/ml), due to varying degrees of patient hydration at presentation. Hence, random measures of urinary PDG demonstrated poor specificity (32.8%) in correctly differentiating normal from abnormal gestations, thus limiting its clinical usefulness.

Initiation of angiogenesis by porcine follicular fluid.

Angiogenesis was observed and measured after injection of porcine follicular fluid into rabbit corneas. A qualitative response (0 to 6+) and quantitative measurement (mm/day) were obtained 9 days after injection. Undiluted porcine follicular fluid stimulated angiogenesis with new blood vessels visible by the third day after injection, extending 2.0 to 3.0 mm into the site of injection from the corneal scleral limbus (1 to 4+) by day 9. Angiogenic activity was consistently found in fractions of porcine follicular fluid which precipitated in 20% to 40% saturated ammonium sulfate. Sephadex gel filtration of the 20% to 40% saturated ammonium sulfate fraction resulted in fractions with molecular weights of 45,000 to 60,000 and less than or equal to 1500 daltons which stimulated angiogenesis. Charcoal treatment of active fractions did not remove angiogenic activity. Angiogenic activity was retained after heating at 56 degrees C for 1 hour but was lost after boiling (20 minutes). Quantitative measurements of chemotaxis with use of Boyden chambers and mitogenesis by means of tritiated thymidine incorporation were performed. Follicular fluid from small follicles contained greater chemotactic activity than follicular fluid from medium or large follicles. The 20% to 40% saturated ammonium sulfate precipitate that eluted through Sephadex G-100 with a molecular weight of 45,000 to 60,000 daltons contained angiogenic, mitogenic, and chemotactic activity. In conclusion, porcine follicular fluid contains angiogenic factors that may be associated with perifollicular neovascularization during folliculogenesis.

Initiation of angiogenesis by human follicular fluid.

Angiogenesis was observed and measured after injection of human follicular fluid into rabbit corneas. Undiluted human follicular fluid stimulated angiogenesis in every case, with new blood vessels visible 3 days after injection and extending 2.0 millimeters from the corneal scleral limbus into the injection site by day 15. Stimulation of angiogenesis was lost by heating or diluting the follicular fluid but was retained after charcoal stripping or dialysis. Human follicular fluid contains an angiogenic factor that may be associated with perifollicular neovascularization during folliculogenesis.