Survival of cryopreservation and thawing with all blastomeres intact identifies multicell embryos with superior frozen embryo transfer outcome.

OBJECTIVE: To assess the impact of survival of cryopreservation and thawing with all blastomeres intact on the outcome of multicell frozen ET. DESIGN: Retrospective study. SETTING: Academic assisted reproductive technology program. PATIENT(S): One hundred sixteen exclusively multicell frozen ETs in 78 patients. INTERVENTION(S): Frozen ET. MAIN OUTCOME MEASURE(S): Relation of embryonic blastomere survival to the outcome of frozen ET (i.e., pregnancy). RESULT(S): When at least one embryo survived with all blastomeres intact, the total pregnancy rate (biochemical, clinical, or delivered) was 37.7%, the clinical pregnancy rate was 24.6%, and the delivered pregnancy rate was 18.8%. When no embryo survived with all blastomeres intact, the corresponding rates were 10.6%, 8.5%, and 6.4%. The differences in the total pregnancy rate and the clinical pregnancy rate were statistically significant. The delivered pregnancy rates approached statistical significance. CONCLUSION(S): Multicell embryonic survival of cryopreservation and thawing with all blastomeres intact identifies embryos with superior developmental potential.

Is there a risk of cytomegalovirus transmission during in vitro fertilization with donated oocytes?

OBJECTIVE: To define the risk of human cytomegalovirus (HCMV) transmission from donated oocytes. DESIGN: Prospective study. SETTING: University IVF program. PATIENT(S): Sixty-seven couples undergoing 72 cycles of IVF-ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum from both partners (women: n = 71; men: n = 60) was obtained for detection of antibodies to HCMV. Semen before preparation (n = 53), sperm after preparation (Percoll gradient; n = 47), cervical mucus aspirated at the time of oocyte aspiration (n = 70), and uninseminated oocytes and embryos not suitable for cryopreservation (n = 568) were frozen in liquid nitrogen. Polymerase chain reaction was used for detection of HCMV (immediate early 1 gene) in all samples collected. RESULT(S): Serum antibodies to HCMV were found in 62% of the women and 37% of the men tested. Human cytomegalovirus DNA was detected in 25% of the ejaculates and in 19% of the cervical mucus samples. There was no amplification of HCMV DNA from oocytes or embryos. CONCLUSION(S): Because we were unable to amplify HCMV DNA from any of the oocytes or embryos, it seems unlikely that HCMV is transmissible through oocyte or embryo donation.

Repeated transmission of X-linked ocular albinism type 1 by a carrier oocyte donor.

OBJECTIVE: To report the transmission of unsuspected X-linked ocular albinism in an oocyte donor program. DESIGN: Case report. SETTING: University medical center. PATIENT(S): A 24-year-old white female oocyte donor and the outcomes of three recipient pregnancies. INTERVENTION(S): Clinical assessment and molecular diagnostic tests. MAIN OUTCOME MEASURE(S): Mutation detection. RESULT(S): Demonstration of carrier status and multiple transmissions of a mutant allele. CONCLUSION(S): We describe the transmission of a mutant allele for X-linked ocular albinism from an unsuspected carrier female oocyte donor to three independent pregnancies. We emphasize the need for diligent inquisition to clarify any unusual history of ocular or constitutional signs that might signify an X-linked disorder.

Endometrial biopsy during hormone replacement cycle in donor oocyte recipients before in vitro fertilization-embryo transfer.

OBJECTIVE: To examine the usefulness of a trial cycle of hormone replacement therapy (HRT) and endometrial biopsy before the actual ET cycle in recipients of donated oocytes. DESIGN: Retrospective review. SETTING: Clinical practice at the South Texas Fertility Center, San Antonio, Texas. PATIENT(S): Thirty-six concurrent patients who underwent a trial cycle of HRT with endometrial biopsy before the ET cycle with donated oocytes fertilized in vitro. INTERVENTION(S): Patients > or =40 years of age received 100 mg of i.m. progesterone in oil daily; patients <40 years of age received 50 mg daily. Endometrial biopsies were performed during the late luteal phase of the trial cycle. MAIN OUTCOME MEASURE(S): Histologic dating of the biopsy specimens was correlated with the chronologic date of the biopsy. RESULT(S): Five of 20 patients > or =40 years of age had out-of-phase biopsies. All 16 patients <40 years of age had in-phase biopsies. All out-of-phase biopsies subsequently were corrected with higher doses of progesterone. Pregnancy rates after fresh and frozen ETs were not significantly different between the two age groups. CONCLUSION(S): Patients > or =40 years of age are at risk of having out-of-phase endometrial biopsies while they are receiving standard HRT despite receiving higher doses of progesterone. Trial HRT cycles with endometrial biopsies are recommended.

Regulation of rat granulosa cell alpha-inhibin expression by luteinizing hormone, estradiol, and progesterone.

OBJECTIVE: Our purpose was to assess the effects of follicle-stimulating hormone, luteinizing hormone, forskolin (an adenylyl cyclase activator), estradiol, and progesterone on alpha-inhibin promoter activity in an in vitro fusion gene-transfection system. STUDY DESIGN: A fusion gene consisting of the alpha-inhibin 5' flanking and promoter regions linked to the chloramphenicol acetyl transferase reporter gene was constructed. A granulosa cell line originally derived from an inbred strain of Berlin Duckrey rats was transiently transfected with the fusion gene. Fusion gene activity was determined by measuring chloramphenicol acetyl transferase activity in transfected cells. RESULTS: Both follicle-stimulating hormone and luteinizing hormone activated the alpha-inhibin promoter. Activity in response to combined luteinizing hormone-forskolin treatment was greater than the summation of the activities of the two treatments individually, suggesting that the effects of luteinizing hormone might be partially mediated by second messengers other than cyclic adenosine monophosphate. Gonadotropin-stimulated activity was diminished by estradiol and combined estradiol-progesterone treatments. CONCLUSIONS: The stimulatory effects of follicle-stimulating hormone and luteinizing hormone on alpha-inhibin production are mediated at least in part by stimulation of the alpha-inhibin promoter. The stimulatory effects are blunted by estradiol-progesterone. These observations may partially explain how alpha-inhibin is down-regulated in vivo in response to the preovulatory luteinizing hormone surge.

Diminished alpha-inhibin messenger ribonucleic acid in in vitro fertilization-embryo transfer poor responders reflects declining follicle reserve.

OBJECTIVES: To quantitate and compare granulosa cell alpha-inhibin messenger RNA (mRNA) levels in IVF-ET poor and good responders and thereby learn how alpha-inhibin mRNA levels change in states of diminished ovarian responsiveness. DESIGN: Ribonucleic acid analysis of stored luteinized granulosa cell samples. SETTING: Academic tertiary care institution. PATIENTS: Fifty-three women undergoing follicle aspiration for IVF-ET were studied. Patients were classified as poor responders (n = 16) or good responders (n = 37) according to their E2 concentration on the day of hCG; the E2 of poor responders was < 1,000 pg/mL (3,671 pmol/L) and that of good responders was > or = 1,000 pg/mL (3,671 pmol/L). MAIN OUTCOME MEASURES: Messenger RNA levels were measured using dot blot RNA analysis. The following parameters were determined or derived: total mRNA levels, total alpha-inhibin mRNA levels, alpha-inhibin mRNA per follicle, and proportional alpha-inhibin mRNA as the ratio of alpha-inhibin mRNA:total mRNA. RESULTS: Proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle were not significantly different between poor responders and good responders. Total mRNA and total alpha-inhibin mRNA levels, however, were diminished significantly in poor responders. CONCLUSIONS: The observations that proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle do not significantly change in poor responders, whereas total alpha-inhibin mRNA does, indicate that the decrease in total alpha-inhibin mRNA in poor responders reflects a decreased pool of total mRNA, likely because of a reduction in follicle number. These findings are in contrast to other recent reports that describe a change in granulosa cell function accompanying states of decreased ovarian responsiveness.

Elevated serum progesterone levels on the day of human chorionic gonadotropin administration in in vitro fertilization cycles do not adversely affect embryo quality.

OBJECTIVE: To assess the effect of an elevated serum P level on the day of hCG administration in an IVF cycle on resulting embryos by evaluating their performance at subsequent frozen ET. DESIGN: A retrospective study. PARTICIPANTS: Ninety-six consecutive patients undergoing frozen ET cycles were studied in a tertiary care center. MAIN OUTCOME MEASURES: Serum obtained on the day of hCG administration in an IVF cycle was assayed for E2 and P by RIA. The main outcome measured was the development of a clinical pregnancy in a subsequent frozen ET cycle. RESULTS: Using a previously described breakpoint in serum P concentration of 0.9 ng/mL (2.86 nmol/L), 8 of 69 (11.6%) frozen ETs in which embryos from low P level IVF cycles were transferred and 7 of 27 (25.9%) frozen ETs of embryos from elevated P level IVF cycles were transferred resulted in the development of clinical pregnancies. Although this does not clearly demonstrate superiority of embryos obtained from elevated P cycles, employing a power calculation, the probability that the pregnancy rate in the elevated serum P group is at least equal to the observed rate in the low P group is 92.8%. CONCLUSION: These data suggest that an elevated serum P level on the day of hCG administration does not adversely affect the quality of oocytes or resulting embryos.

Serum progesterone concentrations on the day after human chorionic gonadotropin administration and progesterone/oocyte ratios predict in vitro fertilization/embryo transfer outcome.

PURPOSE: In gonadotropin-releasing hormone analogue-pretreated in vitro fertilization-embryo transfer cycles, pregnancy rates are inversely related to serum progesterone levels on the day of administration of human chorionic gonadotropin. The relationship of the progesterone concentration on other days in the periovulatory period to pregnancy rates in such cycles is little studied. We therefore retrospectively analyzed the relationship between progesterone concentrations on the day after human chorionic gonadotropin and pregnancy in 114 cycles, 28 and 23 of which produced clinical and ongoing/delivered pregnancies, respectively. To assess the effect of the extent of follicular luteinization on success, we also studied the relationship between the progesterone concentration per oocyte retrieved and pregnancy for the day of and day after human chorionic gonadotropin. RESULTS: Progesterone concentrations on the day after human chorionic gonadotropin were inversely associated with clinical pregnancy by multiple logistic regression analysis (P < 0.05). Progesterone/oocyte ratios were inversely associated with clinical pregnancy (P < 0.05) and ongoing/delivered pregnancy (P < 0.02) for both the day of and the day after human chorionic gonadotropin. CONCLUSION: The study results extend the window of time during which elevated progesterone concentration is associated with poor outcome to at least 2 days. This finding is consistent with hypothetical mechanisms attributing the link between progesterone concentration and outcome to either endometrial or follicle/oocyte events. The association of lack of follicular luteinization (low progesterone per oocyte ratios) and favorable outcome suggests a predominant effect of progesterone on follicle/oocyte quality. Further studies are needed to clarify the mechanisms underlying the association between progesterone and in vitro fertilization-embryo transfer outcome.

Complications associated with the absorption of hysteroscopic fluid media.

OBJECTIVES: To review the literature concerning complications resulting from absorption of hysteroscopic fluid distension media and to describe methods to treat and prevent these complications. DESIGN: All pertinent literature on fluid distension media used for endoscopy, as well as relevant reports concerning the management of fluid and electrolyte imbalance, was reviewed. RESULTS: The absorption of large volumes of electrolyte-free, low-viscosity fluid may result in volume overload with water intoxication. Volume overload may cause pulmonary edema, and water intoxication may lead to hyponatremia, hypo-osmolarity, and cerebral edema. In contrast, the absorption of dextran-70 may cause volume overload secondary to the oncotic effect of intravascular dextran. Dextran-70 has been associated with anaphylaxis and coagulation disorders. TREATMENT: The use of diuretics is advocated. Urine output must be closely monitored. Judicious correction of electrolyte imbalance will prevent morbidity. PREVENTION: Meticulous attention to intraoperative fluid balance is imperative. A multichannel hysteroscope is necessary to keep intrauterine pressure low. Extensive surgical procedures may need to be performed in stages. CONCLUSIONS: Severe volume overload and electrolyte imbalance may result from fluid absorption during operative hysteroscopy. Most complications may be avoided by closely monitoring fluid balance intraoperatively.

Consecutive versus alternating cycles of ovarian stimulation using human menopausal gonadotrophin.

Ovarian stimulation is an effective treatment for patients with ovulatory dysfunction and unexplained infertility. An initial report has suggested that consecutive cycles of ovarian stimulation can be employed without causing a diminished response in the second cycle. However, this observation has neither been confirmed nor has a regimen of consecutive stimulation cycles been compared to one of alternating stimulation cycles. Accordingly, 44 consecutive and 54 alternating cycles of stimulation were evaluated in patients (n = 42) who were treated with human menopausal gonadotrophin (HMG) alone. Human chorionic gonadotrophin (HCG) 10,000 IU was administered i.m. when at least one follicle exceeded 16 mm in mean diameter, and this was followed by either intercourse or intrauterine insemination. Using each patient as her own control, we were unable to demonstrate any differences in mean HMG dose requirements, endocrine parameters or follicular development on the day of HCG administration, or ovulation rates in the second consecutive cycle compared to the second alternating cycle. Clinical pregnancies resulted significantly more often in a consecutive cycle (8/22) than in an alternating cycle (2/27, P = 0.029). We conclude that consecutive cycles of ovarian stimulation with HMG are not detrimental and may, in fact, result in increased cycle fecundity compared to alternating stimulation cycles.

A prospective, randomized trial comparing two different intrauterine insemination regimens in controlled ovarian hyperstimulation cycles.

OBJECTIVE: To compare a single periovulatory intrauterine insemination (IUI) with a regimen employing two IUIs, one before ovulation and one after ovulation, in patients undergoing controlled ovarian hyperstimulation with human menopausal gonadotropins (hMG) combined with human chorionic gonadotropin (hCG). DESIGN: A randomized, prospective trial. PARTICIPANTS: Thirty-one consecutive patients undergoing 49 cycles of controlled ovarian hyperstimulation/IUI were studied in a tertiary care setting. MAIN OUTCOME MEASURES: Ovulation was determined sonographically. The establishment of a clinical pregnancy was defined by either ultrasonographic verification of cardiac activity within an intrauterine fetus, or histologic confirmation of trophoblast in a surgical specimen. RESULTS: Clinical pregnancies developed in 2 of 23 cycles in the single insemination group, compared with 12 of the 23 cycles in the double insemination group. Cycle fecundity was significantly higher for group II (0.522) than for group I (0.087) patients (P = 0.003). CONCLUSION: In hMG/hCG cycles, two IUIs timed as described above are superior to one periovulatory insemination.

Effects of clomiphene citrate and leuprolide acetate on luteal-phase hyperprolactinemia during ovarian stimulation with menopausal gonadotropins.

Hyperprolactinemia, a known modulator of reproductive function, occurs commonly in women undergoing ovarian stimulation with human menopausal gonadotropins (hMG). Clomiphene citrate (CC) and gonadotropin releasing hormone analogues (GnRHa), when administered during the luteal phase, attenuate the hyperprolactinemic response to hMG. We asked whether follicular-phase administration of CC and GnRHa, as employed clinically in women undergoing ovarian stimulation for in vitro fertilization or gamete intrafallopian transfer, would alter the incidence and severity of hMG-induced luteal-phase hyperprolactinemia. Seventy-five percent of all patients had at least one luteal prolactin level greater than 25 ng/ml, and 40% had mean luteal-phase prolactin levels greater than 25 ng/ml. The incidence of hyperprolactinemia was similar in pregnant and nonpregnant cycles. The incidence of hyperprolactinemia was similar for both the GnRH agonist-treated group and those given clomiphene citrate. The increase in mean luteal prolactin levels over the follicular-phase baseline level was significantly greater in the CC-treated group (P = 0.03). This was due to the significant suppression of follicular-phase baseline prolactin levels in patients receiving CC. We conclude that neither CC nor GnRHa administration in the follicular phase prevents luteal-phase hyperprolactinemia in women undergoing ovarian stimulation with hMG.

Serum progesterone levels predict success of in vitro fertilization/embryo transfer in patients stimulated with leuprolide acetate and human menopausal gonadotropins.

Serum progesterone (P4) levels greater than 2.86 nmol/L (0.9 ng/mL) on the day of hCG administration are reportedly associated with decreased pregnancy rates in in vitro fertilization/embryo transfer (IVF/ET) cycles. To further assess this phenomenon we measured serial serum P4, LH, and estradiol levels in 115 consecutive patients undergoing stimulation for IVF/ET with midluteal leuprolide acetate and human menopausal gonadotropins. IVF/ET cycle outcome was retrospectively correlated with P4 levels on the day of hCG administration. Two critical breakpoints were identified, 1.27 nmol/L (0.4 ng/mL) and 286 nmol/L (0.9 ng/mL). Clinical pregnancies occurred in 9 of 18 patients in group I (P4, less than 1.27 nmol/L) compared to 11 of 81 patients in group II (1.27 less than P4 less than 2.86 nmol/L; P = 0.001) and 0 of 14 patients in group III (P4, less than or equal to 2.86 nmol/L) (P = 0.001). Eleven patients in group III had cryopreservation of embryos during that cycle. Six subsequently underwent frozen embryo transfer, and clinical pregnancies occurred in 2, both of whom have delivered. These findings demonstrate that even modest increases in serum P4 levels (greater than 1.27 nmol/L) are associated with reduced pregnancy rates in IVF/ET cycles. In addition, it appears that the mechanism may not exclusively involve poor oocyte quality.

Follicular size at the time of human chorionic gonadotropin administration predicts ovulation outcome in human menopausal gonadotropin-stimulated cycles.

OBJECTIVE: The objectives of this study were: (1) to correlate follicle size by transvaginal sonography with ovulation outcome in cycles of controlled ovarian hyperstimulation with human menopausal gonadotropins; (2) to determine if follicular size on the day of human chorionic gonadotropin (hCG) administration predicts the incidence of ovulation; and, if so, (3) to derive a mathematical model that predicts the number of expected ovulations in any given cycle of controlled ovarian hyperstimulation. DESIGN: A retrospective analysis. PARTICIPANTS: Forty-nine consecutive patients undergoing 122 cycles of controlled ovarian hyperstimulation were studied in a tertiary care setting. MAIN OUTCOME MEASURES: Follicular size and evidence of ovulation were determined sonographically. The main outcome measure was the rate of ovulation per follicle size. RESULTS: The percentages of follicles measuring less than or equal to 14 mm, 15 to 16 mm, 17 to 18 mm, 19 to 20 mm, and greater than 20 mm on the day of hCG administration that subsequently ovulated were 0.5%, 37.4%, 72.5%, 81.2%, and 95.5%, respectively. CONCLUSIONS: (1) Follicular size on the day of hCG administration correlates with the incidence of ovulation. (2) The expected number of ovulations in any given controlled ovarian hyperstimulation cycle can be predicted with 95% confidence using the accompanying equation.

Immediately preovulatory levels of messenger ribonucleic acid for inhibin alpha-subunit are diminished in granulosa cells from successful in-vitro fertilization-embryo transfer.

Inhibin, a gonadal glycoprotein which suppresses pituitary gonadotrophin secretion, preferentially follicle stimulating hormone, has been extensively characterized. It consists of two covalently bound subunits, the alpha- and beta-subunits, encoded by separate genes. In this study, the expression of messenger ribonucleic acid (mRNA) for the inhibin alpha-subunit was studied by Northern blot analysis in granulosa cells of women undergoing in-vitro fertilization/embryo transfer (IVF/ET). Three patient groups were studied: women who failed to become pregnant (n = 11), women who became pregnant but experienced early spontaneous abortion (n = 3) and women who conceived normal ongoing pregnancies (n = 4). Granulosa cells were obtained at the time of follicle aspiration. Levels of alpha-subunit mRNA were 40% lower in patients establishing normal pregnancies than in those who failed to become pregnant or who spontaneously aborted. Thus, a relative diminution of immediately preovulatory levels of mRNA for inhibin alpha-subunit is a marker of success in clinical IVF/ET cycles. This marker of IVF/ET success can be related to previously established markers of success (increased follicular fluid oestradiol and decreased follicular fluid cyclic adenosine monophosphate) by known physiological mechanisms.

Premature luteinizing hormone surges in menopausal gonadotropin-stimulated cycles in monkeys: lack of initiation by progesterone.

The occurrence of spontaneous luteinizing hormone (LH) surges in women receiving human menopausal gonadotropins (hMG) for in vitro fertilization-embryo transfer is a significant clinical problem. One hypothetical mechanism is that premature progesterone (P) secretion occurring in the high estradiol (E2) milieu produced by hMG triggers the spontaneous LH surge. To investigate this possibility, 11 rhesus and cynomolgus monkeys were stimulated with hMG. At maximal ovarian stimulation, monkeys were injected with 15 micrograms/kg P (n = 3), 30 micrograms/kg P (n = 3), or 1,000 IU human chorionic gonadotropin (hCG) (n = 5; controls). Blood for E2, P, and LH was drawn twice daily in the periovulatory period and daily before and after this period. Laparoscopy was performed after P or hCG injection. In the 6 monkeys receiving P, no LH surges were detected. Further, postinjection P profiles and laparoscopy showed no evidence of ovulation. Controls demonstrated laparoscopic and hormonal evidence of ovulation. These findings suggest that P does not trigger LH surges in hMG-stimulated cycles.

Expression of the human inhibin alpha-subunit gene in preovulatory granulosa-theca cells.

Inhibin, a gonadal peptide that suppresses pituitary follicle-stimulating hormone, with lesser or no effect on luteinizing hormone, has recently been purified and the complementary deoxyribonucleic acid sequences cloned. Inhibin contains two subunits, labeled alpha-subunit and beta-subunit. Here we report for the first time the detection of human inhibin alpha-subunit gene expression in preovulatory granulosa-theca cells by Northern analysis. The transcript is the same size as previously reported for human placenta and corpus luteum, suggesting that the same gene is being expressed in all three tissues. These findings are consistent with previously reported Southern analysis of deoxyribonucleic acid, which showed only one copy of the alpha-inhibin gene in the human genome. Thus current data strongly suggest that there is only one copy of the inhibin alpha-subunit gene in the human genome, and this same gene is expressed in granulosa-theca cells, corpus luteum, and placenta.

Gamete intrafallopian transfer vs superovulation with intrauterine insemination for the treatment of infertility.

Superovulation with intrauterine insemination (SO-IUI) has been suggested as an alternative to gamete intrafallopian transfer (GIFT), despite the absence of controlled or comparative trials. We retrospectively analyzed all GIFT and SO-IUI cycles performed concurrently from January 1985 to August of 1987 at a single university center. Pregnancy rates were significantly better for GIFT than SO-IUI (P less than 0.001), with an odds ratio of 3.25 (P = 0.001). Stepwise multiple logistic regression identified factors that correlate with pregnancy: absence of endometriosis (P = 0.05), infertility less than 3 years' duration (P = 0.002), TMS greater than or equal to 30 X 10(6) (P = 0.005), and treatment with GIFT rather than SO-IUI (P = 0.001). These data give a first approximation of the increased efficacy of GIFT versus SO-IUI and provide valuable insight into significant confounding variables to be considered when planning a randomized, prospective trial to evaluate these techniques.