Francisco J. Rojas

Oocyte donation and gamete intrafallopian transfer in premature ovarian failure

Gamete intrafallopian transfer (GIFT) was performed in eight patients with premature ovarian failure (POF), using donated oocytes. The steroid replacement protocol consisted of the administration of increasing dosages of 17β-estradiol (E2) and progesterone (P). Hormonal replacement was maintained until day 100 of gestation. All patients underwent an evaluation cycle in which serum levels of E2 and P were monitored and an endometrial biopsy was performed on day 21 or 22. All cases of GIFT were performed between days 12 and 15. Six clinical pregnancies were achieved in eight cycles (75% success rate). Three patients delivered and three are in their second or third trimester. No ectopics or miscarriages occurred. These results offer a promising approach for the establishment of fertility in agonadal patients.

Status of hCG/LH Receptor and G Proteins in Human Endometrium During Artificial Cycles of Hormone Replacement Therapy

Objectives: We examined the existence of hCG/LH receptors and associated GTP-binding (G) proteins in membrane fractions of nonpregnant human endometrium and investigated whether their expression is affected, in vivo, by estrogen and progesterone replacement therapy. Methods: A pool of normal endometrial biopsy specimens (n = 5) was initially used to characterize receptors and G proteins. Subsequently, biopsy specimens (n = 22) were obtained from 11 patients undergoing evaluation cycles of hormone replacement therapy (HRT). From each patient, two specimens were collected on successive cycle days: on day 0 (last day of estrogen) and on either day 3, 6, or 9 of progesterone supplementation. Both hCG/LH receptor and G proteins were determined in membrane (10,000 × g) fractions by immunoblot analysis using specific polyclonal antibodies against synthetic fragments of hCG/LH receptor and against G proteins. Membrane fractions from rat brain and rat corpus luteum were used as controls. Proteins were loaded on the gel under reducing conditions. Results: The receptor antibody immunoreacted with a protein of approximately 68 kd in endometrial membranes. A similar protein was detected in rat corpus luteum. The G-protein antibodies detected Gsα, Gi3α, Gi1α/Gi2α, and common beta subunits in endometrial membranes with a molecular weight of 48-42 kd, 41 kd, 40 kd, and 37 kd, respectively. Analysis of membranes obtained during HRT indicated that levels of hCG/LH receptors remained fairly constant throughout the cycle days (days 0, 3, 6, and 9). Similar results were observed for Gi1α/Gi2α and Gi3α. In great contrast, Gsα was low at day 0 but increased with the administration of progesterone (days 3, 6, and 9). Conclusions: Human endometrium contains both membrane-bound hCG/LH receptors and associated G proteins. During HRT, progesterone ssupplementation to estrogen therapy enhances the expression of Gsα protein subunit, but not hCG/LH receptors.

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.

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

Penetration of zona-free hamster oocytes using human sperm aspirated from the epididymis of men with congenital absence of the vas deferens: comparison with human in vitro fertilization *

OBJECTIVES To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF). DESIGN Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF. PATIENTS, PARTICIPANTS Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm. RESULTS Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative. CONCLUSIONS These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.

Successful gamete intrafallopian transfer following failed artificial insemination by donor: evidence for a defect in gamete transport? *

Gamete intrafallopian transfer (GIFT) was offered as an alternative treatment to 48 women who failed to conceive after artificial insemination with donor semen (AID) in numerous attempts (9 to 24 cycles). The evaluation of these women showed no major cause of infertility as evidenced by normal endocrine, cervical, uterine, and tubal factor studies. Their partners were either azoospermic or severely oligoasthenospermic. During the GIFT cycle, follicular development was induced with (1) clomiphene citrate (days 3 to 7) plus human menopausal gonadotropins (hMG) from day 6 on or (2) human follicle-stimulating hormone (days 3 to 4) plus hMG (day 5 on), until ultrasound revealed 2 follicles 16 mm and serum estradiol (E2) was greater than 700 pg/ml. Human chorionic gonadotropin (hCG) 10,000 IU was administered, and 36 hours later follicular aspiration was performed. One to three oocytes and 100,000 motile sperm were transferred to each fallopian tube through the fimbria via laparoscopy or minilaparotomy. Twenty-seven clinical pregnancies were achieved (56%) per GIFT cycle. Eight miscarriages occurred during the first trimester (29% of all pregnancies), whereas no ectopic pregnancies were observed. These data conclusively show the value of the GIFT procedure in the treatment of cases with failed AID.

Regulation of gonadotropin-stimulable adenylyl cyclase of the primate corpus luteum☆

In an effort to understand the molecular mechanisms that control luteal function in the human and nonhuman primates, we have investigated the experimental conditions for expression of gonadotropin-induced adenylyl cyclase (AC) in membrane particles from primate corpus luteum (CL) and some of the factors modulating the enzyme activity. We also examined the usefulness of the cell-free model for studying the role of AC in the regulation of CL functions in human and nonhuman primates. Enzyme activity was dependent on guanine nucleotide and Mg ion. Dose-response curves showed that the AC activation constants for hCG was about 0.1 microgram/ml. This value did not shift after the addition of guanine nucleotide. Enzyme responsiveness to prostaglandin E2 was small and, in contrast to a number of other nonprimate species, AC from the human CL was not stimulated by catecholamines. Calcium directly inhibited responsiveness of hCG-sensitive AC; inhibition was significant at 0.5 mM CaCl2 (in the presence of 1 mM EDTA and 2 mM ATP), being 90% at 2.5 mM CaCl2. These results support the concept that Ca2+ might play a role in the regulation of gonadotropin action and life span of human CL. Changes in AC activities during luteal phase and pregnancy were similar in the CL of monkeys and humans. Thus, in both cases, maximal gonadotropin responsiveness was observed during the midluteal phase. Also, during pregnancy (term and early pregnancies), responsiveness to exogenous hCG in vitro was very low, but the enzyme was readily responsive to NaF (10 mM) and forskolin (100 microM). These activities suggest that the tissue remains functionally active during pregnancy. It is concluded that the cell-free AC system is an effective model to study the cellular mechanisms that regulate luteal function in human and nonhuman primates.

The role of the adenylyl cydase system in the regulation of corpus luteum function in the human and in nonhuman primates

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL. In the menstrual cycle, luteal AC responsiveness to LH/hCG was detectable during the midluteal phase, but not during the late luteal phase or in the follicular phase of the following cycle. In addition, nonhormonal stimulation was high in CL obtained during the midluteal and late luteal phases, but declined drastically by the follicular phase of the next cycle. In early pregnancy, the enzyme was unresponsive to LH/hCG stimulation, yet its sensitivity to nonhormonal stimulation was similar, if not identical, to that of midluteal phase CL. Functional activity was also evident at the end of pregnancy. These results demonstrate that expression of AC activity in primate luteal membrane changes significantly with varying hormonal status under physiologic conditions. It is concluded that the AC system in luteal membranes is an effective model to study the mechanisms that regulate function and life span of the human and nonhuman primate CL.

Evidence that hormone receptors couple and activate a common signal transducer adenylyl cyclase in corpus luteum**Presented at the 43rd Annual Meeting of The Pacific Coast Fertility Society, Coronado, California, April 26 to 30, 1995.

OBJECTIVES To investigate whether membrane-bound hormone receptors in corpus luteum (CL) couple to one common adenylyl cyclase (AC) or whether each receptor is coupled to its own AC. DESIGN Plasma membranes from rat CL were used to assess the coupling of hormone receptors to AC under conditions allowing full expression of the enzyme system. The response to hCG, prostaglandin E2 alpha (PGE2), and isoproterenol were analyzed. MAIN OUTCOME MEASURE Adenylyl cyclase activity was monitored by the direct conversion of [infinity-32P]ATP into [32P]cyclic AMP. Results were expressed as pmol/min per mg membrane protein. RESULTS Addition of hCG (200 mIU), PGE2 (100 microM), and isoproterenol(100 microM) in the presence of a saturating (100 microM) concentration of guanine nucleotide resulted in a marked stimulation of the enzyme compared with controls, reaching 552 +/- 28, 537 +/- 42, and 558 +/- 32 (mean +/- SEM), respectively. Addition of hormones in pairs or all three together did not result in an additive response of the individual effects. Thus, in the presence of the three hormones together, AC stimulation was 582 +/- 41 (n = 5), a value that was not significantly different from the stimulation observed with each hormone alone. CONCLUSION These data indicate that luteal membranes contain a single AC system. Therefore, hormone receptors couple and activate a common signal transducer AC in luteal membranes.

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis**Supported in part by a grant from the Memorial Health Services, Department of Obstetrics and Gynecology, University of California, Irvine.

OBJECTIVE To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification. DESIGN PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. RESULTS Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. CONCLUSION These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.