Nitric oxide as a regulator of embryonic development.

The role of nitric oxide (NO) in activation of cGMP is well established. It has been proposed that the ratio of cAMP to cGMP may be important in the regulation of preimplantation embryonic growth and differentiation. Therefore, we determined the ability of murine preimplantation embryos to produce NO. In addition, NO as an endogenous smooth muscle relaxant and vasodilator is a candidate for involvement in embryo implantation because this process requires increased vascular permeability and uterine quiescence at the sites of blastocyst apposition. Nitrite assays, an indirect measure of NO production, indicate that preimplantation murine embryos produce NO. This production was reversibly inhibited by culture of embryos in medium containing a nonspecific NO synthase (NOS) inhibitor (NG-nitro-L-arginine). Additionally, inhibition of normal development was observed in embryos cultured with NOS inhibitor. NO levels increased in culture medium when ovariectomized progesterone-treated animals were exposed to estrogen for 1 h in utero. Such hormonal treatment induces implantation. These data indicate that NO levels are regulated by estrogen and may be important in regulation of implantation. In addition, these data demonstrate for the first time that NO production appears to be required for normal embryonic development.

The prognostic value of serum progesterone and quantitative beta-human chorionic gonadotropin in early human pregnancy.

OBJECTIVE: Our purpose was to determine whether serum progesterone, with or without quantitative beta-human chorionic gonadotropin, is predictive of pregnancy outcome within the first 8 weeks of gestation in asymptomatic women. STUDY DESIGN: Asymptomatic patients at < 8 menstrual weeks' gestation were prospectively evaluated. The enrollment protocol included history, physical examination, ultrasonographic confirmation, and blood sample collection for beta-human chorionic gonadotropin and progesterone. The association between progesterone and beta-human chorionic gonadotropin values and pregnancy outcome was determined by logistic regression analysis. A receiver-operator characteristic curve was generated on the basis of the sensitivity and specificity of progesterone results. RESULTS: Seventy-four patients were evaluated in this study. The mean serum progesterone level for viable pregnancies was 22.1 ng/ml, which was significantly greater than that for the nonviable gestations, 10.1 ng/ml (p < 0.001). A single progesterone level was predictive of pregnancy outcome (p < 0.001). The probability of an abnormal pregnancy outcome with a serum progesterone level < or = 6 ng/ml was 81%. A single beta-human chorionic gonadotropin level did not contribute to the prediction of pregnancy outcome (p = 0.59). CONCLUSIONS: Serum progesterone alone, within the first 8 weeks of gestation, is predictive of pregnancy outcome.

Subclinical pregnancy loss in clomiphene citrate-treated women.

OBJECTIVE: To ascertain the subclinical pregnancy loss rate in clomiphene citrate (CC)-treated infertile women compared with women of normal fertility. DESIGN: Following a prospective format, serum samples were taken during the luteal phase of 92 menstrual cycles associated with CC treatment and 47 cycles in normal women. Human chorionic gonadotropin (hCG) assay sensitivity was 0.25 IU/L. Human chorionic gonadotropin assay was validated against 95 nonpregnant cycles. Criterion for pregnancy was a single serum sample greater than or equal to 0.5 IU/L. SETTING: All subjects were under clinical management at the University of Virginia Health Sciences Center. PATIENTS AND PARTICIPANTS: Patients undergoing CC induction of ovulation with satisfactory ovulatory response were candidates for the study group (n = 34). Control subjects of proven normal fertility were recruited (n = 22). Nonpregnant control subjects were sexually abstinent or had been surgically sterilized (n = 89). INTERVENTION: A serum sample was taken during the late luteal phase of all subjects. RESULTS: Thirteen percent of CC-treated cycles and 4.3% of normal control cycles were subclinical losses (P = 0.09). Fifty percent of CC-induced pregnancies were subclinical losses compared with 16.6% of normal control pregnancies (P = 0.05). Of CC patients who had at least one subclinical loss 47.6% later conceived a term pregnancy compared with 15.3% who did not have a subclinical loss (P = 0.06). CONCLUSION: Subclinical pregnancy loss is more common in CC-treated women than normal women and may be a predictor of subsequent normal conception.

Preservation of pulsatile luteinizing hormone release during postpartum lactational amenorrhea.

To evaluate the pulsatile mode of immunoactive LH release during physiological lactational amenorrhea, we withdrew blood samples at 10-min intervals for 24 h from breastfeeding women (n = 9) at both 3 weeks and 3 months postpartum. Nonlactating women (n = 7) were sampled similarly in the early follicular phase of the normal menstrual cycle. Objective LH pulse analysis revealed that the mean frequencies of pulsatile LH release were similar at both times postpartum and in menstruating young women. By 3 months postpartum, mean serum PRL concentrations had declined 50%, and serum LH peak areas doubled. In contrast, LH interpulse interval, peak duration, and maximal, incremental, and fractional LH pulse amplitude did not change significantly. When deconvolution analysis was used to assess pituitary responses to two pulses of exogenous GnRH at 3 months (vs. 3 weeks) postpartum, we found significant increases in maximal LH secretory rates and the total mass of LH secreted. There was no change in the duration or timing of the evoked LH secretory burst and/or the estimated half-life of endogenous LH. In summary, during lactational amenorrhea, pulsatile LH release occurs at a mean frequency no different from that in the normal early follicular phase. As hyperprolactinemia wanes, there is increased pituitary responsiveness to exogenously administered GnRH and a doubling of spontaneous serum LH concentration peak areas. Such amplitude changes are consistent with the hypothesis of increased endogenous GnRH drive (e.g. augmented GnRH secretion per burst and/or increased pituitary responsiveness to available GnRH) during recovery of the postpartum hypothalamopituitary-ovarian axis.

Dynamics of pulsatile prolactin release during the postpartum lactational period.

To investigate the pulsatile nature of PRL release in the physiologically hyperprolactinemic postpartum period, we sampled blood at 10-min intervals for 24 h in each of 6 healthy lactating women at both 3 weeks and 3 months postpartum. The subsequent immunoactive PRL time series were subjected to episodic peak detection (Cluster analysis) and multiple parameter deconvolution analysis. The 24-h mean serum PRL concentrations were significantly higher at 3 weeks than at 3 months postpartum; viz. 113 +/- 12 vs. 66 +/- 15 micrograms/L (P = 0.003). Assessment of episodic PRL pulsatility revealed significantly higher maximal PRL peak heights (296 +/- 63 vs. 141 +/- 44 micrograms/L), fractional peak heights (863 +/- 150 vs. 374 +/- 58%), incremental peak amplitudes (250 +/- 60 vs. 96 +/- 3 micrograms/L), and peak areas (13 +/- 3 vs. 4 +/- 1 mg/L.min) in the earlier postpartum period. In contrast, PRL peak frequencies and interpulse intervals were not different in the early and late postpartum sessions. Deconvolution analysis revealed that the mean mass of PRL secretory bursts was significantly greater at 3 weeks (182 +/- 4.1 micrograms/L) than 3 months (15 +/- 1.6 micrograms/L). There were no changes in the calculated half-life of endogenous PRL viz. 29 +/- 2.5 min (3 weeks) vs. 26 +/- 3.0 min (3 months). We conclude that physiological postpartum hyperprolactinemia is achieved by selectively altering the endogenous secretory rate in each PRL release episode, with no change in the number of bursts of PRL discharged or the PRL half-life.

Oocyte retention after follicle luteinization.

Indirect evidence supports the existence of the luteinized unruptured follicle syndrome in infertile women. To seek direct evidence of oocyte retention, infertile and normal women were studied in the early and midluteal phase by visual documentation of ovulation stigma, needle aspiration of ovarian follicles, and peritoneal fluid collection for estradiol and progesterone assay. Luteal phase was confirmed by endometrial biopsy (postovulation day 2 to 8). In normal control subjects (n = 16), 25% of test cycles were stigma-negative and no oocytes were recovered. In infertile group (n = 23), 43% of test cycles were stigma-negative. Five oocytes were recovered including one from a stigma-bearing follicle. Peritoneal fluid steroid levels failed to discriminate stigma-positive from stigma-negative cycles in either group. Oocyte retention after luteinization occurs in infertile women.

Exogenous estrogen therapy for treatment of clomiphene citrate-induced cervical mucus abnormalities: is it effective?

Clomiphene citrate (CC) may have an adverse effect on cervical mucus (CM) quality and quantity. A placebo-controlled study was performed to assess the effect of exogenous follicular phase estrogen (E) on CM. Subjects qualified for inclusion by repeated demonstration of poor CM while on CC therapy as judged by spinnbarkeit, quantity, and viscosity. Subjects were treated by a randomized, placebo-controlled format using: (1) oral micronized estradiol (E2), 2 mg; (2) conjugated Es, 5 mg, or (3) placebo administered on cycle days 9 to 14. Cervical mucus was scored blindly during therapy within 48 hours before ovulation. Twelve subjects were observed through 36 treatment cycles with mean (+/- SD) CM scores: micronized E2, 4.2 +/- 1.8; conjugated Es, 4.3 +/- 1.7; and placebo, 4.7 +/- 2.9. There was no significant difference in mean values (P = 0.96, analysis of variance) or frequency of CM score greater than 4 (P = 0.85, Fisher exact test). We conclude that therapy with the E preparations tested did not improve the quality or quantity of CM in CC-treated patients.

Vaginal sonography findings and hCG dynamics of early intrauterine and tubal pregnancies.

Early intrauterine gestational sac recognition by ultrasound is useful for discriminating between intrauterine and tubal pregnancies. Transvaginal ultrasound offers improved resolution in the imaging of the uterus and adnexa. Seventy-four women with normal intrauterine pregnancies, 18 women with spontaneous abortions, and 34 women with tubal pregnancies were evaluated by transvaginal ultrasound and serial hCG determinations. Intrauterine pregnancies were followed within the first 50 days of gestation. In women with a normal uterus and singleton pregnancy, sacs were consistently recognized in association with an hCG level greater than 2004 mIU/mL (First International Reference Preparation). Two-to three-millimeter sacs were recognized in association with an hCG level of 2032 +/- 1253 on cycle day 36.1 +/- 2.0. When sacs were less than 5 mm in mean diameter, 33% exhibited a prominent echogenic rim, 38% exhibited a partial echogenic rim, and 29% exhibited no echogenic rim. Fetal cardiac activity was recognized in association with an hCG level of 27,284 +/- 11,479 on cycle day 45.0 +/- 2.9. The hCG doubling time was 1.46 +/- 1.0 days. Apparent loss of multiple gestational sacs was observed in five normal pregnancies. Apparent bleeding in the intrauterine cavity was observed in eight patients and associated with a 38% abortion rate. One false-positive sac was observed among 34 tubal pregnancies (3%). The extrauterine sonographic finding of a solid adnexal mass or cul-de-sac fluid was specific but not sensitive for the diagnosis of tubal pregnancy. The hCG doubling time in tubal pregnancies was 7.69 +/- 9.8 days. Transvaginal ultrasound and serial hCG determinations are useful for the early recognition of tubal pregnancy.

Subsequent fertility in women who undergo cardiac surgery.

A retrospective review was undertaken on all pediatric and reproductive-aged females who underwent cardiac surgery and required cardiopulmonary bypass from 1958 through 1986. The purpose of this study was to define the fertility of these patients after surgery and to compare their reproductive performance with that in the general population. Analysis was complete for 208 patients. These patients make up the following cardiac surgical categories: septal defect repairs, 92; commissurotomies, 60; valve replacements, 46; tetralogy of Fallot repairs, 6; and coronary artery bypass procedures, 4. Of 208 patients, 68 (32.7%) attempted pregnancy after surgery. Infertility was defined in five women as follows: endometriosis, 3; ovulatory dysfunction, 1; unknown, 1. A total of 64 patients conceived 121 pregnancies with the following outcomes: live births, 98; spontaneous abortion, 9; ectopic pregnancy, 2; therapeutic abortion, 12. Our results suggest that infertility is not an apparent disorder after cardiac surgery and that subsequent pregnancy outcomes are similar to those in the general population.

Acute bilateral diffuse pulmonary shadowing after evacuation of hydatid mole.

Acute bilateral diffuse pulmonary shadowing complicates the resection of hydatid mole approximately 10% of the time. Etiologic factors include trophoblastic emboli, pulmonary emboli, high output congestive failure, and DIC. Prompt recognition and emergency oxygen therapy with PEEP are imperative for survival.

Intravasation during hysterosalpingography using oil-base contrast medium--a second look.

Hysterosalpingography is a study performed commonly to assess the upper reproductive tract in infertile women. A risk with hysterosalpingography is either lymphatic and/or venous intravasation with or without embolization. Of 593 consecutive hysterosalpingograms performed with a low-viscosity oil-base medium, intravasation occurred in 41 cases (6.9%). Embolization was documented in six patients. There were no recognized adverse sequelae from either intravasation or embolization. Intravasation was frequently associated with pathology of the upper genital tract, and suspected tubal occlusion was the most common abnormality detected. Proper timing of the procedure and avoidance of excessive instillation pressure will diminish the incidence of this complication. The use of both a low-viscosity oil-base medium and high-resolution fluoroscopic image intensification may increase the detection of intravasation.

Protein synthesis and secretion in human decidua of early pregnancy.

Proteins synthesized and secreted by first trimester decidua in primary culture were identified. Explants were cultured for 24 h, in RPMI-1640 or Dulbecco's Modified Eagles Medium containing the radioactive amino acid 35S-methionine or 3H-proline. Electron microscopy of explants before and after 24 h of culture demonstrated the relative purity of the decidua, maintenance of cell integrity, and ultrastructural features indicative of active protein synthesis and secretion. Proteins synthesized and secreted by the explants into the medium were analyzed by fluorography of one-dimensional polyacrylamide gels in the presence of sodium dodecyl sulfate. By comparison of radiolabeled proteins from four women, eight 35S-methionine-labeled bands of 78, 70, 60, 50, 43, 34, 25, and 23 kDa were identified as common decidual peptides. A comparison of autoradiographs of the medium from decidual cultures to decidual cell homogenates showed that seven of these peptides were enriched in the culture medium. When labeled peptides from fibroblast cultures were compared to labeled proteins from decidual cultures each of the common decidual peptides (except the 70 kDa protein) occurred only in the decidual culture medium. Comparison of 3H-proline and 35S-methionine-labeled decidual proteins revealed that the 78, 70, 60, 50, and 34 kDa proteins were of similar fluorographic intensity when labeled with the two different amino acids. The 43, 25, and 23 kDa proteins appeared to contain more methionine, and proteins at 36, 20, 13, and 12 kDa were proline-rich, but contained less methionine. The seven decidual explant-specific, 35S-methionine-labeled secreted proteins were concentrated and purified by preparative gel electrophoresis, and antisera were generated to four of the putative decidual secretory proteins.

Utility of the 24-hour delay hysterosalpingogram film.

Controversy exists regarding the ideal contrast media for hysterosalpingography. A unique property of oil-base contrast media is the availability of a 24-hour delay radiograph for further assessment of tubal patency and adhesions. A review was undertaken of the delay films in 131 cases performed by use of oil-base contrast media with subsequent surgical confirmation of pelvic findings. A 97% predictive accuracy was achieved with regard to distal obstruction and a 79% accuracy with regard to pelvic adhesions. Objective criteria for the evaluation of 24-hour delay hysterogram films were developed and are illustrated.

Surgical management of distal tubal occlusion.

During the 9-year period ending April, 1985, 103 women had bilateral and unilateral cuff salpingostomy at the University of Virginia Hospital. Currently accepted principles of microsurgery were used as well as an "antiadhesion" regimen consisting of intravenous dexamethasone and antibiotics, intraperitoneal dextran, and postoperative hydrotubations. The postoperative tubal patency rate was 75.6% in tested patients (91.2% in patients known to have conceived). Forty women (38.8%) conceived and 26 (25.2%) have had one or more term pregnancies. Fourteen women (13.5%) had ectopic pregnancies but three of these have also had term pregnancies. Fourteen women (13.5%) had first-trimester abortions. Two of nine women who had repeat salpingostomies have carried pregnancies to term as have two of 10 women who had ampullary salpingostomies. The extent of tubal disease remains the single most important factor with regard to subsequent successful pregnancy.

Quantitative effects of intraperitoneal 32% dextran 70 on serum immunoglobulin levels in women undergoing gynecologic surgery.

The use of intraperitoneal 32% dextran 70 results in quantitative postoperative changes in human IgG and IgA levels that are statistically different from the alterations induced by the surgical procedure alone.

Homologous insemination--revisited.

Between July 1, 1969, and December 31, 1983, 1774 patients were evaluated in the private infertility clinic at the University of Virginia Hospital. Homologous artificial insemination was performed in 158 patients for the following indications: male factor (75.3%), cervical factor (16.7%), sperm deposition problem (3.7%), patient's request (2.5%), and immunologic factor (1.8%). Whole-ejaculate insemination was performed in 68 patients; split ejaculates were used in 90 women. Additional fertility factors in 140 patients included endometriosis, anatomic abnormalities, and ovulatory difficulties. Of the 158 women, 23 (14.6%) had a total of 27 pregnancies (mean number of cycles, 5.7); 135 failed to conceive during treatment with homologous artificial insemination (mean number of cycles, 8.4). Most pregnancies (70.4%) occurred in those patients in whom homologous artificial insemination was used for a male factor. Among the 135 couples who failed to conceive with homologous artificial insemination, 20 (14.8%) patients subsequently had 25 pregnancies. Male factor had been the indication for homologous artificial insemination in 72% of the couples. Pregnancy outcomes were similar in all subgroups. Homologous artificial insemination may not be indicated for male factor-related infertility.

Reproductive performance of patients treated with clomiphene citrate.

From 1967-1980, 488 infertile women underwent induction of ovulation with clomiphene citrate at the University of Virginia Hospital. Conception occurred during treatment cycles in 209 women (group 1), but 279 women (group 2) failed to conceive. The outcome of pregnancy was defined in group 1, and the pregnancies that occurred in both groups after treatment was discontinued were analyzed. Early pregnancy wastage (spontaneous abortion and ectopic pregnancy) in these groups was compared with the accepted incidence in the general population. Although the rates of adverse pregnancy outcome were higher in our study populations, there was no statistical difference when compared to the general population.