The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: a randomized study.

PURPOSE: The aim of this study was to examine the influence of endometrial cells on the fertilization rate and early embryonic morphology following routine in vitro fertilization (IVF). Cryopreservation with subsequent thawing allowed the use of autologous somatic cells, thus minimizing the risk of transmission of infective agents. Interpatient variability was eliminated by randomizing oocytes from each cycle into the control or coculture group. RESULTS: Two hundred ninety-four oocytes from 24 IVF cycles (21 patients) were included in the study (145 coculture and 149 control). The normal fertilization rate of control oocytes (56.4%) was not significantly different from that of oocytes cocultured with endometrial cells (61.4%). The mean number of blastomeres in cocultured embryos (3.65) was not significantly different from the number in control embryos (3.46) 2 days after insemination, but the proportion of embryos with minimal or no fragmentation was significantly higher in the coculture group [34/84 (40.5%) vs. 17/80 (21.3%); P < 0.01]. CONCLUSIONS: The inclusion of cryopreserved autologous endometrial cells in routine clinical IVF procedures does not influence fertilization or the early cleavage rate but may reduce the extent of embryo fragmentation during the early cleavage divisions.

Culture of marmoset blastocysts on matrigel: a model of differentiation during the implantation period.

BACKGROUND: The objective was to develop an experimental model for studying the differentiation of trophoblast and inner cell mass (ICM) during the early stages of implantation in primates. METHODS: Marmoset monkey blastocytes were used in these studies. Ovulation was timed by plasma progesterone assays in ovarian cycles initiated by administering a luteolytic agent to mating marmosets. Embryos were recovered from the uterus usually at the eight-cell stage and cultured in minimum essential medium containing fetal calf serum, insulin, and transferrin. The embryos that formed hatched blastocysts by about day 11 after ovulation were transferred for further development in Matrigel-coated culture chambers. After 2, 4, and 6 days of development, two blastocysts were processed at each interval and serially sectioned for light and electron microscopy. RESULTS: All blastocysts adhered to the Matrigel at their embryonic pole within 24 hours. Adherent polar cytotrophoblast was differentiating to syncytiotrophoblast at all time intervals, but syncytium was not detected in mural trophoblast until day 4 after attachment. By day 2 syncytial microvilli and processes had penetrated the Matrigel surface, whereas by days 4 and 6 cytotrophoblast that was differentiating to syncytiotrophoblast had invaded the matrix. Since all blastocysts maintained their structural integrity progressive differentiation of the ICM, endoderm and presumptive mesoderm was observed. A small amniotic cavity was observed at 2 days and by 6 days a distinct cavity separated polarized epiblast and amnion cells. Visceral and parietal endoderm were present at 2 days, and a completed primary yolk sac was observed by 4 days after attachment. In all blastocysts a basal lamina lined the inner surface of mural and polar trophoblast and the basal surface of the differentiating ICM. CONCLUSIONS: The developmental time sequence of the cultured blastocysts closely resembled the time frame reported for marmoset embryos implanting in utero. An effective model for studying trophoblast invasion and differentiation of embryonic germ cell layers has been established.

Characterization of large luteal cells and their secretory granules during the estrous cycle of the cow.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.

Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species.

Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivites as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny.

Hormonal changes during luteal regression in farmed fallow deer, Dama dama.

Concentrations of progesterone, oxytocin and PGFM (pulmonary metabolite of PGF-2 alpha) were measured in plasma from peripheral blood samples collected from 5 fallow does every hour or 2 h for 12-h periods on Days 15-20 inclusive of the oestrous cycle (i.e. luteolysis). For 3 does that exhibited oestrus on Day 21, plasma progesterone concentrations fluctuated between 3 and 10 ng/ml on Days 15-18 inclusive. Thereafter, values declined progressively to attain minimum concentrations of less than 0.05 ng/ml on Day 20. Basal concentrations of plasma oxytocin and PGFM fluctuated between 5 and 20 pg/ml and 10 and 100 pg/ml respectively. Episodic pulses of plasma oxytocin (greater than 300 pg/ml) occurred on Days 15 and 16, whereas pulses of plasma PGFM (greater than 400 pg/ml) occurred on Days 19 and 20. There was little apparent correlation between episodic pulses of the two hormones. For 2 does that exhibited oestrus on Day 22, plasma progesterone concentrations declined to minimum values of 1.0-1.5 ng/ml by Day 20. One of these does showed very high levels of oxytocin secretion throughout the sampling period while the other showed an apparent paucity of oxytocin secretory periods. Two does hysterectomized on Day 13 of their second oestrous cycle failed to exhibit further oestrous cycles. Continual elevation of plasma progesterone concentrations (2-6 ng/ml) for an 8-month period indicated persistence of the corpus luteum after hysterectomy. It is concluded that luteolysis in fallow deer involves episodic secretion of both oxytocin and PGF-2 alpha.

Arginine vasopressin and oxytocin in the porcine corpus luteum.

The relationship between ovarian oxytocin (OT), vasopressin and progesterone, was determined in porcine corpora lutea collected at various stages of the estrous cycle. Fractionation by reverse phase high performance liquid chromatography (HPLC) of extracts from mid-cycle corpora lutea showed the presence of arginine vasopressin-like (AVP) material, but not lysine vasopressin-like (LVP) activity. The tissue content of progesterone reached maximal amounts at mid-luteal phase, and decreased rapidly at the end of the cycle. However, tissue concentration of progesterone was the highest in early luteal phase. OT content was unchanged throughout the luteal phase of the cycle but declined at the end of the cycle. Tissue concentration of OT decreased from early in the cycle but was still measurable in the corpus albicans. Changes of AVP content and concentration were substantially the same as that of OT, except for a significant increase in AVP concentrations at the end of the cycle. These data confirm the presence of OT, indicate the dominant presence of AVP and suggest that both hormones may be involved in porcine CL function.

Characterization of neurophysin in the anterior pituitary gland of sheep.

Portions of sheep anterior pituitary lobe tissue were extracted under acid conditions and assayed for the two neurophysins (oN-III and oN-I/II) by radioimmunoassay. In all tissues examined, oN-III and oN-I/II immunoreactivities were detected. Using a combination of isoelectric focusing and polyacrylamide gel electrophoresis, the neurophysins of the anterior pituitary gland behaved like oN-III and oN-I/II. oN-III of the anterior pituitary was purified by affinity chromatography and high-performance liquid chromatography. This corticotroph oN possessed an amino acid analysis similar to that of oN-III and an N-terminal amino acid sequence, including residues 1-24, identical to that of authentic oN-III. These findings support the work of others who have identified neurohypophysial hormones in the anterior pituitary gland.

Effect of systemic intravenous infusion of PGF-2 alpha and 13,14-dihydro-15-keto-PGF-2 alpha on the release of oxytocin-associated neurophysin from the ovary in the ewe.

Systemic intravenous infusion of physiological concentrations of PGF-2 alpha and its major metabolite, 13, 14-dihydro-15-keto-PGF-2 alpha (PGFM) into non-pregnant ewes possessing a corpus luteum induced the release of oxytocin-neurophysin. These results suggest that, during luteolysis, endogenous release of uterine PGF-2 alpha would be able to stimulate the release of ovarian oxytocin and oxytocin-neurophysin from the ovary.

Levels of oxytocin-like activity and progesterone in follicular fluid from in vitro fertilization cycles.

Oxytocin-like immunoreactivity, estradiol, and progesterone were measured in follicular fluid collected during oocyte collection in an in vitro fertilization program in which clomiphene citrate was used to stimulate follicular development. Follicles which yielded morphologically normal embryos after fertilization of the oocyte had oxytocin concentrations ranging from less than 10 to 600 ng/liter. Oxytocin concentrations did not differ between follicles from 12 pregnancy cycles (median, 169; N = 21) and follicles from 12 nonpregnancy cycles (median, 110; N = 18). Oxytocin concentrations were correlated negatively with progesterone concentrations (Spearman's rank correlation coefficient r = -0.50; P = 0.001). In cycles with some follicles having progesterone concentrations less than 10 and some greater than 10 mumol/liter, oxytocin concentrations were higher in the less progestogenic follicles in 15 of 16 cases.

Effect of systemic or intrauterine injections of indomethacin on plasma oxytocin-neurophysin concentrations in ewes over the time of normal corpus luteum regression.

This study was undertaken to investigate the effect of systemic or intrauterine injections of indomethacin, a known prostaglandin (PG) synthetase inhibitor, on peripheral plasma oxytocin-associated neurophysin (OT-N) concentrations in ewes over the time of expected luteolysis. In the first experiment, 9 ewes were given i.m. injections of indomethacin (4 mg/kg live weight, n = 4) or vehicle (n = 5) 3 times/day over Days 13-15 of the estrous cycle. Blood samples were collected at hourly intervals from 0700 h on Day 13 to 1800 h on Day 15 post-estrus. In the second experiment, indomethacin (20 mg, n = 5) or the injection vehicle (n = 4) was given twice daily into the uterine horn over Days 12-14 post-estrus. Blood samples were collected at hourly intervals from Day 12 to 14. In the third experiment, 4 additional ewes were bled at 5-min intervals from 1200 to 1600 h on Day 13 of the estrous cycle. Plasma samples were analyzed for OT-N and 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) to provide an indirect index for ovarian oxytocin and uterine prostaglandin F2 alpha release, respectively. Results from the first experiment indicated that surges in plasma OT-N concentrations occurred in the vehicle-treated ewes but were suppressed in ewes given systemic injections of indomethacin. Intrauterine indomethacin injections did not cause a significant reduction in the maximum peak height or number of peaks when compared with the control ewes. In the third experiment, there was a marked increase in plasma OT-N concentrations, but no significant rise in plasma PGFM concentrations in one ewe.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin, oxytocin-associated neurophysin, and prostaglandin F2 alpha concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep.

Oxytocin, oxytocin-associated neurophysin (neurophysin), prostaglandin F2 alpha (PGF2 alpha), and progesterone concentrations were measured in the utero-ovarian vein (UOV) of sheep during the estrous cycle and early pregnancy. On days 13-16 of the cycle, large pulses of PGF2 alpha, oxytocin, and neurophysin were measured in samples collected at hourly intervals from the UOV draining a corpus luteum (UOV/CL). Most of the PGF2 alpha pulses (96.5%) coincided with a pulse of both oxytocin and neurophysin, whereas only 55.6% of oxytocin pulses coincided with a pulse of PGF2 alpha. Therefore, during luteolysis in sheep, uterine PGF2 alpha release is closely associated with ovarian oxytocin release, and oxytocin release is unlikely to be dependent upon a uterine PGF2 alpha stimulus. During frequent sampling, coincident oxytocin pulses were measured in 1) both UOVs when a CL was present in both ovaries and 2) the jugular vein, carotid artery, and UOV/CL, with a significantly higher oxytocin pulse concentration occurring in jugular venous compared with carotid arterial plasma. Pituitary and luteal release of oxytocin may, therefore, occur simultaneously and be controlled by a circulating factor in sheep. Compared to days 13-16 of the cycle, significantly (P less than 0.001) fewer pulses of PGF2 alpha, which were significantly (P less than 0.001) smaller in amplitude, were measured in UOV samples collected frequently during early pregnancy. The frequency of oxytocin pulses observed in the UOV/CL of pregnant sheep was not significantly (P greater than 0.1) different from that observed in cyclic ewes, although most (86.4%) oxytocin pulses occurred in the absence of a PGF2 alpha pulse. In contrast, when a pulse of PGF2 alpha was observed in the UOV/CL of pregnant ewes, it usually coincided with a pulse of oxytocin. The suppression of uterine PGF2 alpha release during early pregnancy is not considered to result from a lack of stimulation by oxytocin.

HPLC separation of vasopressin-like hormones in chicken neurohypophysial extracts.

HPLC techniques were used to identify peptides that possess vasopressin-like immunoreactivity in the chicken neurohypophysis. The presence of arginine vasotocin (AVT) was confirmed together with arginine vasopressin (AVP). That the presence of AVP may be confined to the chicken, and not other avian species, was concluded from the absence of the hormone in the neurohypophysis of the duck and turkey. The chicken thus resembles some members of the suiformes and metatheria in possessing two vasopressin-like peptides.

Non-coordinate localization of corticotroph-neurophysin and beta-endorphin in the anterior pituitary gland of the rat.

Anti-neurophysin serum was applied in the immunohistochemical technique to anterior pituitary tissues obtained from normal and chronically dehydrated rats and also from rats with chronic diabetes insipidus (Brattleboro strain). In all cases there was a positive staining in the corticotrophs, which also stained for either beta-endorphin (beta-END) or adrenocorticotrophin hormone (ACTH). It was concluded that corticotroph-neurophysin may be synthesized independently of either ACTH or beta-END.

Use of a cross-species immunohistochemical procedure to demonstrate the presence of neurophysin antibodies in the serum of a patient treated with Pitressin.

Serum from a patient who had been treated with Pitressin for extended periods was evaluated for the presence of autoantibodies. When this immune serum was used with either an immunofluorescence or an heterologous immunoperoxidase technique, positive staining was observed in the neurophysin-containing cells of the rat supraoptic, paraventricular and suprachiasmatic nucleus. Whereas the immune serum may also be expected to contain antibodies against oxytocin, vasopressin and the anterior pituitary hormones, this could not be substantiated with the immunohistochemical procedures.

Isolation and sequence analysis of oxytocin from the sheep corpus luteum.

Studies have revealed that the corpus luteum (CL) of the sheep releases oxytocin (OT) -like immunoreactivity under normal and physiological conditions. We have now purified and completely characterized the OT-like species from ovine CL and established by Edman degradation and comparative reverse-phase HPLC its identity with hypothalamic oxytocin. On the basis of radioimmunoassay, the characterized oxytocin was the only peptide possessing OT-like immunoreactivity. This study represents the first identification by sequence analysis of oxytocin outside the central nervous system.

Evidence for the pulsatile release of PGF-2 alpha inducing the release of ovarian oxytocin during luteolysis in the ewe.

Frequent blood samples were removed from a utero-ovarian vein, a jugular vein and a femoral artery of 5 ewes during luteolysis. Analysis of these samples for oxytocin-associated neurophysin revealed a significant venous-arterial difference across the ovary and uterus but not across the head. This occurred during the pulsatile surges as well as when levels were basal and confirms the corpus luteum as a major source of the pulsatile surges of oxytocin-associated neurophysin and oxytocin that occur during CL regression and also of the elevated luteal phase concentrations of both hormones. The pulsatile surges of oxytocin-associated neurophysin measured in the utero-ovarian vein were accompanied by the release of an approximately equimolar amount of oxytocin. The concentration of PGF-2 alpha in the utero-ovarian vein samples began to increase before the levels of oxytocin and oxytocin-associated neurophysin started to increase. This suggests that uterine PGF-2 alpha initiates the release of ovarian oxytocin and oxytocin-associated neurophysin during luteolysis in the ewe.

Immunochemical recognition of oxytocin by antiserum to L-prolyl-leucyl-glycine amide.

Antisera were raised in rabbits against Pro-Leu-Gly-NH2 (PLG)-bovine thyroglobulin conjugates. Specificity of the antisera towards oxytocin was evaluated by a radioimmunoassay as well as by immunohistochemical procedures on hypothalamic tissue and hormone bound to Sepharose beads. It was concluded that immunization with PLG can raise antisera that are specific to oxytocin.

Effect of oxytocin on plasma concentrations of 13,14-dihydro-15-keto prostaglandin F and the oxytocin-associated neurophysin during the estrous cycle and early pregnancy in the ewe.

This study was undertaken to determine the effect of exogenous oxytocin on plasma concentrations of the prostaglandin (PG) F metabolite 13,14-dihydro-15-keto-PGF (PGFM) and the oxytocin-associated neurophysin (OT-N) during the estrous cycle and early pregnancy in the ewe. Ewes were given oxytocin (250 mU, i.v.) on Days 3 (n = 4), 8 (n = 5), 13 (n = 4) or 14 (n = 5) of the estrous cycle, and a further 6 ewes were injected on Days 13 (n = 2) and 14 (n = 4) of pregnancy. No significant rises in plasma concentrations of PGFM were observed on Days 3 and 8 of the estrous cycle and on Days 13 and 14 of pregnancy. A marked increase in plasma PGFM concentrations occurred on Day 14 of the estrous cycle with the PGFM levels rising from a mean basal value of 120 pg/ml to a mean maximum value of 415 pg/ml within 2-10 min of administering oxytocin (P less than 0.001). No increases in plasma OT-N concentrations were found in early pregnancy and only 1 of 4 ewes at Day 14 of the cycle showed any significant increase in OT-N concentrations. It is concluded that there is an increase in the responsiveness of the uterine-PGF secretory system to oxytocin during the latter stages of the estrous cycle. During early pregnancy this response was blocked by the presence of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of neurophysins by the ovary in sheep.

Measurements of venoarterial concentration differences across the ovary in anesthetized sheep have demonstrated that the ovary secretes ovine neurophysin I/II (oNP I/II) and that this process is stimulated by the prostaglandin F2 alpha analogue, cloprostenol. A parallel increase in the secretion of oxytocin (OT) was observed in response to cloprostenol, and the mean molar ratio of oNP I/II to OT secreted was 1.2. There was no detectable ovarian secretion of oNP III. Secretion of oNP I/II and OT was absent after hysterectomy. The data support other evidence indicating that the corpus luteum synthesizes OT, and confirm that the neurophysin associated with OT in the sheep is oNP I/II.

Immunohistochemical localization of neurophysin and oxytocin in the sheep corpora lutea.

Antisera raised against neurophysin, oxytocin and vasopressin were used, in conjunction with the immunofluorescence procedure, to localize the neuropeptides in corpora luteal tissue collected from sheep at estrus. Specific immunostaining for neurophysin and oxytocin was observed in all the giant cells of the corpora lutea. In the series of corpora lutea examined staining for vasopressin was not observed.