Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Effects of hCG on prostaglandin synthesis and function of corpus luteum.

The effect of intramuscular injections of human chorionic gonadotropin (hCG) on subsequent in vitro prostaglandin (PG) production by the corpus luteum was studied in the rhesus monkey. Four monkeys received increasing doses of hCG from days 6 to 10 after ovulation. On day 11, laparotomy and luteectomy were performed. Five untreated animals served as controls. The production of prostaglandins F (PGF) and E (PGE) by the corpus luteum of the animals treated wit hCG was significantly lower than that of the controls (P less than .01). After hCG treatment, the decrease in PGF production was greater than that of PGE, resulting in a lower ratio of PGF:PGE production than in the controls (P less than .01). Histologic evaluation of the corpora lutea revealed regressive changes in the control group, whereas signs of active secretion were observed in the hCG-treated group. Progesterone concentrations in peripheral blood of the hCG-treated group were approximately fourfold higher than in the controls, and the hCG-treated group did not have a postmidluteal phase decrease. These results suggest that the ratio of production of PGF to PGE by the corpus luteum of the rhesus monkey may play a role in the control of its lifespan and steroidogenic capacity.

Anatomy and pathology of tubal pregnancy.

The prevalence of tubal pregnancy has increased markedly during the past decade. The reasons for this are obscure. A systematic gross and histopathologic study of 25 consecutive ectopic pregnancies has been performed using a clearing method not used previously for this purpose. In addition, the presence of the corpus luteum and its location in reference to the tubal pregnancy are documented. Results indicate that trophoblastic spread was predominantly intraluminal in 67% of cases. Intratubal hemorrhage, generally in parallel to trophoblastic spread, often led to marked tubal destruction. Histologic evidence of salpingitis was noted in only seven of 24 specimens (29%). The corpus luteum was contralateral to the ectopic pregnancy in five of 21 cases (23.8%). Clinical correlates and areas of future research are discussed. Results indicate that segmental resection of the tubal pregnancy is appropriate in selected cases.

Pattern and duration of ovum transport in the baboon (Papio anubis).

The normal pattern and duration of ovum transport was determined in a group of unmated, spontaneously ovulating baboons (Papio anubis) in which the time of ovulation had been determined directly using serial laparoscopy or indirectly using periovulatory estrogen values, perineal sex skin changes, and corpus luteum histology. At laparotomy, the reproductive tract was segmentally flushed at 24, 48, or 72 hours after ovulation and the position of the ova within the tract ascertained. Ova were consistently recovered from the ampulla 24 hours after ovulation, from both the ampulla and isthmus at 48 hours, and from the uterus 72 hours after ovulation. These results suggest that the baboon constitutes a potentially valuable animal model for the investigation and preclinical evaluation of contraceptive technics affecting tubal function.

Healing and restoration of fertility following microsurgical transection and anastomosis of the rabbit oviduct.

The postoperative time course of healing following tubal microsurgery was examined in the rabbit by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and was correlated with changes in fertility. Groups of animals were bred or used for morphologic studies on days 1, 2, 3, 4, 7, and 14 after surgery. During the first 4 postoperative days, a major loss of ciliary and secretory activity occurred and was characterized by disruption of subcellular organelles. Tissue damage was restricted to within several millimeters of the anastomosis. Healing was well advanced by day 7 and was largely complete by day 14. Corresponding changes in fertility were observed. A significant decline in fertility was noted on the first postoperative day. Thereafter, fertility progressively improved and was normal by day 14.

A new method of transcervical female sterilization: preliminary results in rabbits.

In search of an outpatient procedure for female sterilization, the authors performed the following animal study. Two groups of seven rabbits were subjected to laparotomy, hysterotomy, and hysteroscopy. The uterotubal junction (UTJ) was destroyed with bipolar electrical current, and a plug (Aqualloy, Drachten, The Netherlands) was inserted. The plug on the right side contained quinacrine (Q) in group A and platelet extract (PE) in group B. Histologic assessment by serial sections indicated occlusion of the UTJ in all but one case (96% success rate). This one failure is attributed to the technical difficulties associated with hysteroscopy in the rabbit. The authors project that this method may have the potential of becoming an acceptable outpatient procedure for female sterilization in the human.

Fertility following microsurgical removal of the ampullary-isthmic junction in rabbits.

The role of the ampullary-isthmic junction (AIJ) in fertility was investigated in the rabbit by using microsurgical tuboplastic techniques. Microsurgical transection or resection of the AIJ failed to alter fertility significantly. Nine of ten rabbits which underwent unilateral transection of the AIJ became pregnant on the operated side and eight of the ten became pregnant on the unoperated control side. Of the ten rabbits which underwent unilateral AIJ resection, all became pregnant bilaterally. The results of this study indicate that the AIJ is not necessary for normal fertility in the rabbit.

Fertility and ovum transport after microsurgical removal of the uterotubal junction in rabbits.

The role of the uterotubal junction (UTJ) in fertility and ovum transport was investigated following unilateral microsurgical resection of the UTJ in 15 rabbits. Fourteen animals became pregnant on the UTJ resected side and 14 on the control side following artificial insemination and induction of ovulation. Fifty-two of 61 ova (85.2%) became implanted on the control side, and 56 of 68 ova (82.3%) became implanted on the resected side. Ten days after delivery, artificial insemination and induction of ovulation were repeated. Ovum transport was examined at 60, 66, and 72 hours. There was no significant difference in ovum transport between UTJ resected and control sides. The distribution of ova within the oviduct and the time of their entrance into the uterus was comparable in both sides. These results indicate that the UTJ is not necessary for normal fertility and ovum transport in the rabbit.

Rabbit endosalpinx inhibits implantation in vitro.

OBJECTIVE: To evaluate the ability of rabbit endometrial or endosalpingeal cells to support implantation in vitro and to assess the effects of endosalpinx and endometrium-conditioned media (CM) on blastocyst-endometrial cell interaction. DESIGN: In one experiment, rabbit blastocysts were co-cultured in vitro with endometrial or endosalpingeal cells growing on Matrigel-coated plastic culture plates or Millicell-HA inserts. In a second experiment, rabbit blastocysts were co-cultured with endometrial cells in the presence of fresh medium or of endosalpinx- or endometrial-CM. After 48 or 72 hours attachment to the cell monolayer was evaluated. RESULTS: Blastocysts in co-culture attached to endometrial but not to endosalpingeal monolayers. The addition of CM from cultured endosalpinx significantly decreased embryo attachment to endometrial cells in culture. CONCLUSIONS: These findings in vitro agree with the observation that rabbit endosalpinx in vivo does not support embryo implantation and support the hypothesis that rabbit endosalpinx secretes a factor that prevent tubal implantation.

Endosurgical electrocoagulation of the mesovarium as an alternative method of inducing surgical menopause in monkeys.

This study in the primate demonstrates that electrocoagulation of the mesovarium under endoscopic visualization could successfully replace conventional surgical excision of the ovary.

Comparison of two techniques of suturing in microsurgical anastomosis of the rabbit oviduct.

Microsurgical reconstruction of the rabbit oviduct was undertaken utilizing either through-and-through sutures including the mucosa, or sutures penetrating the serosa and myosalpinx but not the mucosa. Patency and pregnancy rates did not seem to vary with the suturing technique. However, scanning electron microscopic observation revealed abnormal mucosal fold patterns at 3 weeks after surgery. Fibrinous exudates over the surface of the intraluminal sutures increased with time until the entire surface was thickly veiled by 9 weeks. The observation also revealed regeneration of epithelial cells along the suture. These cells included many abnormal forms such as giant cells and misshapen cells. The majority of these were nonciliated. In theory, the intraluminal suture could form a nidus for epithelial hyperplasia which might cause future tubal obstruction. However, the patency and pregnancy rates obtained with through-and-through suturing were similar to those obtained when the endosalpinx was excluded, both in this study and in previous studies from these laboratories.

Electrophysiology of the rabbit oviduct following tubal microsurgery.

The ability of tuboplastic microsurgery to restore electrical continuity across an anastomosis site was investigated in the rabbit. Three to six weeks following transection and end-to-end anastomosis of the tubal ampulla or isthmus, the pattern of electrical activity adjacent to and across the anastomosis site was examined. Recordings were made in vitro using arrays of closely spaced suction electrodes 68 to 72 hours after an ovulating dose of human chorionic gonadotropin. The pattern of electrical activity across anastomosis sites was not significantly different from corresponding portions of unoperated control oviducts. Microsurgical anastomosis restored electrical continuity between anastomosed segments in both the ampulla and isthmus.

PIP: This study evaluates the efficiency of tubal microsurgery in restoring tubal functional continuity across an anastomosis site. 11 adult virgin female New Zealand white rabbits were divided into 2 groups: one group of 5 rabbits had their oviducts unilaterally transected at the isthmus level and reanastomosed under microscopic control; the other group of 6 rabbits had their oviducts transected at the ampulla level and reanostomosed similarly. Contralateral unoperated oviducts of the animals were used as controls. The pattern of electrical activity adjacent to and across the anastomosis site was evaluated 3 to 6 weeks following transection and end-to-end anastomosis of the tubal ampulla or isthmus. Arrays of closely spaced suction electrodes were recorded in vitro 68 to 72 hours after an ovulating dose of human chorionic gonadotropins was administered. Consistent electrical continuity was observed following anastomosis of the ampulla and isthmus; no significant differences in the overall pattern of electrical activity in anastomosed sites or corresponding areas of unoperated control tubes from within the same animal at any postsurgical interval (P 0.05) were observed. The findings provide support to the usefulness of microsurgery in sterilization reversal attempts.

Pelvic adhesions following microsurgical and macrosurgical wedge resection of the ovaries.

The incidence of postoperative adhesion formation following microsurgical and macrosurgical ovarian wedge resection was contrasted in 10 adult female rhesus monkeys. Bilateral wedge resection was performed on day 10 of the luteal phase using microsurgical technique on one ovary and macrosurgical technique contralaterally. Animals were examined 4 weeks postoperatively. Adhesion formation occurred in only one ovary in which microsurgery had been employed (10%). In contrast, adhesion formation followed macroscopic ovarian wedge resection in five ovaries (50%). All adhesions were periovarian, emanating from the suture line on the ovarian surface. Adhesions were most common on the nonovulatory ovary.

In vitro maturation of baboon oocytes retrieved at the time of cesarean section.

OBJECTIVE: To investigate the feasibility of oocyte retrieval at the time of cesarean delivery and the potential of such oocytes to undergo nuclear maturation in vitro using a baboon model and an established culture system. DESIGN: Randomized, controlled animal study. SETTING: Research foundation and university research laboratory. ANIMAL(S): Mature pregnant baboons. INTERVENTION(S): In vitro culture of aspirated oocytes with or without epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Oocyte yield, germinal vesicle breakdown, polar body extrusion. RESULT(S): A total of 246 oocytes were retrieved (mean, 35; range, 14-67). Eighty-seven oocytes (35%) underwent germinal vesicle breakdown and 72 oocytes (29%) extruded a polar body. A chi2 analysis revealed no significant effect of EGF on outcome parameters. No effect of gestational age or maternal age on oocyte yield or development was observed. CONCLUSION(S): A sizeable proportion of oocytes obtained from puerperal primates exhibited the capacity to undergo nuclear maturation in vitro.

Rabbit endosalpinx suppresses ectopic implantation.

This study was undertaken to elucidate the mechanism underlying the absence of oviductal ectopic pregnancies in infraprimates. Endosalpingeal circumferential grafts were substituted for endometrium in a group of rabbits. The endosalpingeal grafts interfered with implantation as evidenced by four observations: (1) The nidation indices were lowered, (2) no implantations occurred on the grafted endosalpinx, (3) unattached blastocysts were found in animals grafted with endosalpinx, and (4) the implantation sites were significantly smaller in uterine horns containing endosalpingeal grafts. These findings suggest the presence of a factor in rabbit endosalpinx that actively suppresses ectopic implantation in the oviduct.

Impact of implementation of an embryo storage fee on embryo disposal activity.

OBJECTIVE: To examine the impact of implementation of a new fee for continued storage of cryopreserved embryos on the rate of requests for disposal of embryos. DESIGN: Retrospective cohort study. SETTING: A university-based assisted reproduction program. PATIENT(S): All patients with cryopreserved embryos. INTERVENTION(S): Implementation of a semiannual embryo storage fee of $100 to cover administrative and laboratory costs. MAIN OUTCOME MEASURE(S): The number of embryo disposal requests before and after implementation of the embryo storage fee was compared in relation to the activity of the cryopreserved embryo program as measured by number of frozen embryo transfers. RESULT(S): Annual requests for embryo disposal from 1992 through 1997 ranged from zero to three, which represented 0-5% of the annual frozen embryo program activity. In contrast, a significantly higher number of disposal requests (10, representing 18% of program activity) were received in 1998. CONCLUSION(S): Fees for storage of cryopreserved embryos seem to influence patients' decisions about disposal of cryopreserved embryos.

Fertility following fimbriectomy and tubo-ovarian microsurgery in the rabbit.

It is generally believed that tubal fimbriae function as a highly specialized and indispensable component of the ovum pickup mechanism. The present study was undertaken to examine the feasibility of creating a functional tubo-ovarian relationship following fimbriectomy. Fourteen New Zealand White rabbits underwent unilateral microsurgical fimbriectomy followed by creation of a distal ampullary flap which was oversewn to the exposed ovarian cortex. Two weeks after surgery, the animals were inseminated and induced to ovulate with an intravenous injection of 100 IU of human chorionic gonadotropin. Thirteen of fourteen uterine horns on the control side and ten of fourteen on the operated side became pregnant. The ratios of ova ovulated to those which implanted were 73.2% on the control side and 37.8% on the operated side. Morphologic studies revealed a normal complement of ciliated and secretory cells lining the endosalpinx of the juxtaposed ampullary flap. The portion of ovarian cortex covered by the flap similarly exhibited no morphologic abnormalities. These findings indicate that fertility can be maintained in the absence of fimbriae and suggest a surgical technique for restoration of fertility following elective sterilization by fimbriectomy or following pathologically induced destruction of the fimbriae.

Deciliation in the puerperal fallopian tube.

Tubal segments obtained from patients at cesarean section and at intervals during the first 5 postpartum days were examined to evaluate puerperal changes in the tubal epithelium. The specimens of tubal epithelium were examined under the scanning and transmission electron microscopes. Ciliated cells were most densely distributed on the fimbria and in the ampulla, and were relatively sparsely distributed in the isthmus. Progressive diminution of numbers of ciliated cells and deciliated of individual cells were noted in specimens obtained during the puerperium. Nonciliated cells were in the resting stage at term pregnancy. Secretory activity returned during the puerperium.

Inhibition of the postcastration rise of luteinizing hormone and follicle-stimulating hormone in female rhesus monkeys (Macaca mulatta) by the administration of a luteinizing hormone-releasing hormone inhibitory analog ([N-Ac-D-Trp1-3,D-p-Cl-phe2,D-Phe6,D-Ala10]-LH-RH).

Regularly cycling rhesus monkeys were bilaterally oophorectomized for study of postcastration rise of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The animals were divided in two groups, control animals, which received vehicle, and experimental animals, which received intramuscularly 1 mg of a potent luteinizing hormone-releasing hormone (LHRH) inhibitory analog ([N-Ac-D-Trp1-3,D-p-Cl-phe2,D-Phe6,D-Ala10]-LH-RH) from the day of castration for 10 days. The controls showed significant elevations of FSH and LH 3 to 4 days after castration, but in the experimental animals the rise in gonadotropins was blocked until the LHRH antagonist administration was discontinued. The dynamics of gonadotropin elevation after the discontinuation of [N-Ac-D-Trp1-3,D-p-Cl-phe2,D-Phe6,D-Ala10]-LH-RH administration were similar to those observed in control animals after castration. The availability of a compound that selectively inhibits FSH and LH secretion in primates opens a new approach to contraception and for the treatment of conditions in which gonadotropin inhibition is desired.

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.