Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils.

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.

Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms.

The molecular weight of the purple, iron-containing glycoprotein uteroferrin has been determined to be 35140 +/- 1610 by sedimentation equilibrium centrifugation. This is consistent with measurements carried out by other procedures. The dry weight of a solution of uteroferrin with an absorption of 1.00 cm -1 at 280 nm was 0.87 mg/ml. Highly purified uteroferrin has a ratio of absorbance at 280 nm to 545 nm of about 13.2 and a revised extinction coefficient at 545 nm of 3.1 . 10(3) M -1 is presented. The iron content of uteroferrin has been determined both colorimetrically and by atomic absorption spectroscopy. One iron atom is present per polypeptide chain. Reduction with dithionite leads to loss of iron, allowing the apoprotein to be prepared. Enzymatic activity can be restored to the apoprotein with Fe(III) or with Cu(II). The copper enzyme has no visible color and has a higher pH optimum than the Fe-uteroferrin. Whereas the phosphatase activity of the latter is increased several-fold by beta-mercaptoethanol, reduction inactivates Cu-uteroferrin. Both forms of uteroferrin have similar Km values for p--nitrophenyl phosphate and are inhibited by molybdate but not by tartrate. Excess Cu(II) and Fe(III) strongly inhibit uteroferrin phosphatase activity and these results may explain the failure of other to restore activity to the apoprotein using Cu(II) and Fe(III) salts.

Components in seminal plasma regulating sperm transport and elimination.

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.

Identification of a novel family of growth hormone-related proteins secreted by rat placenta.

Mid-to-late gestation rat placenta synthesizes a number of proteins related to prolactin, including rat placental lactogen II (rPL-II), rat prolactin-like protein A (rPLP-A) and rat prolactin-like protein B (rPLP-B). This study identifies a new family of proteins synthesized and secreted by gestation day 15 placental explants which exhibit amino acid homology to growth hormone precursors from several species. Placental explant medium was fractionated by ammonium sulfate precipitation and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis to isolate four distinct proteins with Mr values of 28,000, 23,000, 25,000 and 30,000 and pI values of 5.7, 5.7, 5.4 and 5.3, respectively. These proteins represent a major fraction of secretory proteins with Mr in the 20,000 to 30,000 range. Immunoblot analysis showed that none of the four proteins crossreacted with antipeptide antisera against rPL-II, rPLP-A, or rPLP-B. These proteins were electrophoretically transferred from two-dimensional SDS-polyacrylamide gels onto an Immobilon PVDF membrane and N-terminal amino acid microsequencing carried out with a gas phase sequencer. N-terminal sequences of 45, 37, 37 and 32 amino acid residues were identified for proteins 1, 2, 3 and 4, respectively. The four proteins exhibit 76% to 97% homology. Computer analysis further revealed a 28% identity in a 32 amino acid overlap which begins at residue 14 of the 28,000 Mr protein (protein 1) and at residue 31 of growth hormone precursors of rat, mouse and human. The 32 amino acid overlap is 78% homologous if conservative amino acid replacements are included.

In vitro synthesis of oviductal proteins associated with estrus and 17 beta-estradiol-treated ovariectomized ewes.

The objectives of this study were: 1) to identify and characterize ewe oviductal secretory proteins (OSP) de novo synthesized during the estrous cycle; 2) to examine the effect of estrogen (E) and progesterone (P) on synthesis of OSP; and 3) to determine synthesis of OSP by ampulla (A) and isthmus (I). Oviducts were collected from cyclic ewes on days 1, 8, and 16 and ovariectomized (OVX) ewes after treatment with corn oil (CO), E, P, and E + P. Tissue was incubated in the presence of 3H-leucine or 3H-glucosamine. Rates of 3H-leucine incorporation into nondialyzable macromolecules by explants was greater (P less than 0.05) on day 1 than days 8 and 16, greater (P = 0.01) in OVX E-treated ewes, greater in A than I regardless of treatment and greater (P less than 0.05) in A than I with E. Culture medium was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and fluorography. Two major basic proteins (Mr 90-92,000) and three minor proteins were only present on day 1 of estrus. E treatment demonstrated these same proteins which were not found with CO or P treatment. Two-dimensional analysis of medium from A and I cultured separately indicated that A synthesized the two major basic and three minor E-dependent proteins. Of three protein complexes present regardless of treatment or cycle day, two were dominant in the A and one was dominant in the I suggesting a synthetic gradient. Tunicamycin did not inhibit incorporation of 3H-glucosamine into E-dependent proteins. Gel filtration revealed a high Mr fraction (2 x 10(6] 2.5-fold greater on day 1 than day 8 and 2.2-fold greater with E than CO treatment. This study clearly demonstrates: 1) that the oviduct from estrus and E-treated ewes synthesizes two major basic and three minor glycoproteins not detectable on other cycle days or with OVX or P treatment; 2) the E-dependent glycoproteins are synthesized by the A; and 3) a high Mr fraction is induced or amplified by E.

Developmental expression of rat insulin-like growth factor binding protein-2 by astrocytic glial cells in culture.

Brain astrocytes were established in primary culture from postnatal and adult rats to characterize the developmental expression of secreted proteins. Astrocytes cultured from 21-day rat brain, but not 1-day rat brain, secreted a distinct group of proteins with Mr of 35,000 as determined by analysis of [35S]methionine-labeled proteins using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis of this protein group showed 100% identity to rat insulin-like growth factor binding protein-2 (rIGFBP-2), the BRL-3A IGFBP purified from a fetal rat liver cell line. An antiserum was generated against this astrocyte 35,000 Mr protein, and immunoblot analysis revealed a dramatic increase in rIGFBP-2 secretion in astrocytes cultured from 14-day, 21-day, and adult rat brain compared to astrocytes from 1-day and 7-day rat brain. Similar analysis of neonatal rat brain neurons in culture failed to show immunoreactive rIGFBP-2 in cell lysates or secreted protein. Ligand Western blot analysis demonstrated [125I]IGF-II binding to a single protein band which comigrated with a prominant rIGFBP-2 immunoreactive species in nonreduced conditioned medium from 21-day astrocytes. In comparison [125I]IGF-II binding proteins were detected only at low levels in medium from astrocytes cultured from 1-day rat brain and were undetectable in neuron-conditioned media. Northern blot analysis using a rIGFBP-2 complementary DNA revealed 5-fold greater messenger RNA levels in astrocytes from 21-day rat brain compared with astrocytes from 1-day brain, whereas neonate neurons showed no transcripts. Thus, rIGFBP-2 exhibits a pattern of developmental and cell-specific expression in cultured rat brain cells.

De novo synthesis and secretion of prolactin-like protein-B by rat placental explants.

Mid- to late-gestation rat placenta expresses three PRL-related mRNAs, rat placental lactogen-II (rPL-II), rat PRL-like protein-A (rPLP-A), and rat PRL-like protein-B (rPLP-B). The protein product of rPL-II mRNA has been characterized, and the protein products of the rPLP-A mRNA were recently identified. The mol wt of a nonsecreted nonglycosylated rPLP-B protein would be 27,145 based on the mRNA sequence. The present study is the first to report the identification of the rPLP-B protein. Antiserum was generated against a chemically synthesized oligopeptide inferred from a specific region of the rPLP-B cDNA. Three or four distinct proteins synthesized and secreted by rat basal zone explants (day 15 gestation) showed cross-reactivity with the rPLP-B antiserum. The relative mol wt of these immunoreactive proteins is approximately 30,000, with a pI varying from 6.1-6.6. De novo synthesized rPLP-B proteins were not secreted by the explant tissue in the presence of tunicamycin, suggesting that the proteins are glycosylated. These data are consistent with the presence of one potential N-glycosylation site derived from the rPLP-B mRNA sequence. The rPLP-B antiserum showed no cross-reactivity with proteins identified using antisera against rPLP-A, rPL-II, or human pregnancy-specific beta 1-glycoprotein. Immunocytochemical studies were carried out using paraffin sections from placentas of day 14 and 17 pregnant rats which were treated with anti-rPLP-B, followed by avidin-biotin-peroxidase complex. These experiments show perinuclear staining, which was localized in basophilic cytotrophoblast cells, confirming previous in situ mRNA hybridization studies. Although no physiological role has been established for rPLP-B, the synthesis and secretion of this protein by cells in contact only with maternal circulation suggest a hormonal role.

Identification of proteins immunochemically related to human pregnancy-specific beta 1-glycoprotein in the rat placenta.

Human pregnancy-specific beta 1-glycoprotein (hPS beta G) consists of a set of glycoproteins present in placenta and maternal serum. This study characterized proteins in rat placenta that show immunological cross-reactivity with antisera to hPS beta G. Immunocytochemical studies using two independent preparations of anti-hPS beta G showed intense specific staining within basophilic cytotrophoblast cells of the basal zone of the gestation day 15 rat placenta. In contrast, basophilic cytotrophoblasts located in the labyrinth did not stain. Subsequent experiments used gel electrophoresis and immunoblot analysis to compare PS beta G in human placenta and serum with immunoreactive proteins in rat placenta and serum. A set of two or three proteins was detected in human villous tissue and pregnancy serum with apparent mol wt (Mr) ranging from 54,000-76,000. In contrast rat placenta showed a major immunoreactive protein with 120,000 Mr, while rat serum contained bands of 48,000 64,000 and 69,000 Mr. Explant cultures of rat basal zone tissue secreted two [35S]methionine-labeled proteins that were immunoreactive, a major 120,000 Mr species and a minor 76,000 Mr form, with pI values of 4.6-5.5; tunicamycin inhibited the secretion of both species. Thus, a 120,000 Mr glycoprotein appears to be the major tissue and secreted form of rat PS beta G analog in day 15 placenta. Finally, the cytochemical localization of PS beta G-like proteins in rat placenta showed a progressive gestational shift from giant trophoblast cells in the parietal yolk sac placenta on day 12 to the basal zone cytotrophoblast cells by day 15. Data indicate that the pregnant rat may provide an animal model for investigation of the biological function of PS beta G during late gestation.

Insulin receptors in circulating erythrocytes and monocytes from women on oral contraceptives or pregnant women near term.

Altered carbohydrate metabolism occurs in women during pregnancy and in those using oral contraceptives (OC). Insulin binding to circulating erythrocytes and monocytes was studied in 77 nonobese, healthy women volunteers; they were divided into 4 groups: 1) late pregnant (n = 19),2) OC users taking 50 microgram estrogen daily (OC-50; n = 19),3) OC users taking 35 microgram estrogen daily (OC-35; n = 18), and 4) a control group (n = 21). All nonpregnant volunteers were in the luteal phase (days 18-21) of the menstrual cycle. Oral glucose tolerance tests were normal in all groups. Fasting plasma insulin was higher (P < 0.001) in the pregnant group, and plasma insulin responses to the oral glucose tolerance test were higher (P < 0.05) in the pregnant, OC-35, and OC-50 groups compared to that in the control group. The percentage of specific binding of [125I]insulin to 1.2 x 10(7) monocytes/ml (and 4.4 x 10(9) erythrocytes/ml) was similar in all groups (mean +/- SE): pregnant, 6.85 +/- 0.48% (6.85 +/- 0.59%); OC-35, (6.85 +/- 0.40%); OC-50, 6.95 +/- 0.55% (6.73 +/- 0.59%); and control 6.66 +/- 0.64% (7.17 +/- 0.44%). No correlation was found between insulin binding to erythrocytes and monocytes. Average affinity profiles and binding sites per cell (70/erythrocyte and 50,000/monocyte, respesctively) were similar in all groups. Since insulin binding to monocytes in decreased during the secretory phase of the menstrual cycle, one could extrapolate from our data that pregnant women will have lower insulin binding compared to nonpregnant women in the proliferative phase of the cycle; such a report has appeared recently (Beck-Nielsen et al., J Clin Endocrinol Metab 49: 810, 1979). Differences in plasma levels of estrogen and progesterone between the secretory and proliferative phases of the cycle are much smaller than between the nonpregnant state and late pregnancy. Therefore, it remains to be seen whether these steroid hormones would cause, by the same mechanism, a decrease in insulin binding (and insulin resistance) during late pregnancy and in the secretory phase of the cycle.

PIP: This study of altered carbohydrate metabolism in pregnant women and oral contraceptive (OC) users measured insulin binding to circulating erythrocytes and monocytes in 77 nonobese healthy women volunteers. The 77 women were divided into 4 groups: 1) third trimester pregnant (n=19); 2) OC users using 50 mcg estrogen formulations (n=19; OC50); 3) OC users with 35 mcg formulations (n=18; OC35); and 4) controls (n=21). Nonpregnant participants in the study were between menstrual cycle Days 18 and 21 (luteal phase). Oral glucose tolerance tests were performed on all groups, and results were normal for all 4 groups. When fasting plasma insulin was tested, it was higher (P .001) in the pregnant group, and plasma insulin responses to oral glucose tolerance test were higher (p .05) in the pregnant, OC35, and OC 50 groups compared with controls. Specific binding of tritiated insulin to monocytes showed similar percentage in all 4 groups. There was no correlation between insulin binding to erythrocytes and monocytes. The average affinity profiles and binding sites per cell (70/erythrocyte and 50,000/monocyte) were similar in all groups as well.

A function of the allantoic sac in the metabolism of uteroferrin and maternal iron by the fetal pig.

The possible roles of the allantoic sac in metabolism of uteroferrin, the iron-containing, purple phosphatase, were examined. This protein, which originates in the maternal endometrium, was measured in allantoic fluid by a specific double-antibody radioimmunoassay and by its beta-mercaptoethanol-activated acid phosphatase activity. Total uteroferrin reaches a maximum between days 60 and 75 of pregnancy, when it constitutes up to one-third of the total protein, and then declines towards term. Fluid volume, total protein and total iron also reach a maximum around day 60. Two-dimensional polyacrylamide-gel electrophoresis was employed to identify proteins in allantoic fluid. Of the proteins detected, at least three basic and two acidic polypeptides are also characteristic of maternal uterine secretions. The remaining proteins appeared identical to those found in fetal serum and included transferrin, albumin and alpha-fetoprotein. Although uteroferrin acid phosphatase activity is stable when incubated in buffer, the enzyme loses activity in sterile allantoic fluid collected at all stages of pregnancy. Experiments with 59Fe-uteroferrin have shown that allantoic fluid promotes iron loss from uteroferrin and that the metal appears in transferrin. Thus the allantoic sac may serve not only as a depot for uteroferrin accumulation but as a site of active iron metabolism.

Expression and characterization of recombinant rat placental prolactin-like protein C.

Prolactin-like protein C (PLP-C) is a member of the rat placental family of proteins which are structurally related to pituitary prolactin (PRL). In an effort to characterize the receptor specificity and biological activity of PLP-C, we used a PLP cDNA to express the recombinant protein in a bacterial system. The PLP-C cDNA was modified by oligonucleotide mutagenesis and ligated into a human carbonic anhydrase II (hCAII) expression vector. Following a single step affinity purification, the hCAII-PLP-C fusion protein was digested with enterokinase to release a 25 kDa protein. N-Terminal sequence analysis of the 25 kDa band demonstrated identity with PLP-C. A polyclonal antiserum to the fusion protein cross reacted with seven major proteins in rat placental culture media of which two were the native forms of PLP-C. Recombinant PLP-C was not mitogenic in the Nb2 lymphoma bioassay and did not exhibit high affinity binding to rat PRL receptor. The choice of hCA-II fusion allows for rapid purification of rPLP-C which will aid in further investigation of the biological role of PLP-C.

Biological activities of glycosylated and nonglycosylated porcine prolactin.

Nonglycosylated and glycosylated porcine prolactin (PRL) were separated using concanavalin A-Sepharose CL-6B column chromatography and tested for mitogenic and lactogenic activities, as well as immunoaffinity and receptor binding characteristics compared to total (nonseparated) porcine PRL. Mitogenic activity, using Nb2 lymphoma cells, was 4- and 50-fold greater (P less than 0.01) for total PRL than nonglycosylated and glycosylated PRL, respectively. Glycosylated PRL had 64% higher (P less than 0.05) lactogenic activity than nonglycosylated or total PRL. In a homologous radioimmunoassay (RIA), displacement was greatest for total, followed by the nonglycosylated and glycosylated forms of PRL. Competitive inhibition of porcine [125I]-(total) PRL by radioinert total, nonglycosylated and glycosylated PRL in a homologous radioreceptor assay (RRA) indicated similar Ka values for total and nonglycosylated PRL, but different receptor numbers, while radioinert glycosylated PRL had a higher Ka, but bound fewer receptors. Therefore, glycosylated porcine PRL has greater lactogenic activity and higher binding affinity despite decreased mitogenicity, while nonglycosylated PRL had characteristics similar to total PRL. Results from the homologous RRA and the Nb2 assay suggest that both forms of PRL are necessary to achieve biological effects similar to those for total PRL. The two forms of PRL may have individual and collective effects, while changes in the ratio between these forms may influence physiologically diverse effects of PRL on target tissues.

The effects of two years of mestranol treatment on carbohydrate metabolism.

Twenty-nine menstrual age women who had a hysterectomy and oophorectomy were treated cyclically with 80 micrograms of mestranol per day for 2 yr. Their carbohydrate metabolism was evaluated prospectively by doing a 3 hr oral glucose tolerance test after a 100 g glucose load and measuring both blood glucose and plasma insulin levels. The tests were performed before drug treatment and after 2 yr of drug use. Those women that had a "normal" predrug test had some carbohydrate metabolism changes at the 2 yr test with significant elevations of the fasting, 1 and 2 hr blood glucose values, and also significant elevations of the 2 and 3 hr plasma insulin values. Those women with a "borderline abnormal" predrug test had no significant change in either parameter of carbohydrate metabolism however a trend toward similar changes was noted.

PIP: 29 menstrual age women who had hysterectomy and oophorectomy were treated cyclically with 80 mcg of mestranol/day for 2 years. Their carbohydrate metabolism was evaluated prospectively by doing a 3 hour oral glucose tolerance test after a 100 g glucose load and measuring both blood glucose and plasma insulin levels. Tests were performed before a drug treatment and after 2 years of drug usage. Those women who had normal predrug tests had some carbohydrate metabolism changes at the 2 year test with significant elevations of the fasting, 1, and 2 hour blood glucose values, and also significant elevations of the 2 and 3 hour plasma insulin values. Those women with a borderline abnormal predrug test had no significant change in either parameter of carbohydrate metabolism; however, a trend toward similar changes was noted.

Glucagon, insulin and glucose levels in maternal and imbilical cord plasma with studies of placental transfer.

Plasma immunoreactive glucagon, as well as insulin and glucose levels, was measured in 62 women and their infants following a term gestation vaginal delivery. Simultaneously obtained samples were drawn from the maternal antecubital vein (MV), umbilical vein (UV), and umbilical artery (UA). Forty-seven of these subjects were untreated (control) and 15 had received a maternal intravenous injection of 1 mg of glucagon within 40 minutes of delivery. It was shown that the umbilical cord glucagon levels were not different from the maternal levels in the control subjects (mean MU, 181.0; UU, 191.9; UA, 161.0 pg/ml). There was no correlation between the maternal and umbilical glucagon levels or the UV glucagon levels and the insulin or glucose concentrations. Neither the fetal sex, placental weight, or infant weight were correlated with the MV or UV glucagon concentration. Following the glucagon injection, the maternal plasma glucagon levels rose significantly, whereas the umbilical blood values did not change. These results suggest that glucagon does not significantly pass through the human term placenta.

Human placental lactogen levels in multiple pregnancies.

Serum human placental lactogen (hPL) levels were measured in duplicate with a radioimmunoassay in 206 serum samples at 30 and 36 weeks' gestation from women with normal singleton pregnancies (75) or pregnancies with twins (37). One triplet pregnancy was also studied. The results show a significant elevation of hPL in the women with twin pregnancies at both the 30th (7.0 vs 6.0 microgram/ml) and the 36th (9.2 vs 7.4 microgram/ml) weeks. One-third of the twin pregnancies had values of hPL in excess of 8.0 microgram/ml at 30 weeks and more than half had values in excess of 9.0 microgram/ml at 36 weeks. The triplet pregnancy had an hPL value of 11.0 microgram/ml at 36 weeks' gestation. These data support the potential usefulness of serum hPL measurements in the screening profile for the detection of high-risk pregnancies.

Carbohydrate metabolism in women with a twin pregnancy.

Carbohydrate metabolism was evaluated in 24 women with a twin pregnancy and 24 women with a singleton pregnancy. The groups were of similar age, parity, weight, and gestational age. In each woman an intravenous glucose tolerance test was done using a 25-g glucose load in the last half of gestation. Both blood glucose and plasma insulin levels were measured and statistically compared. The plasma human placental lactogen levels were significantly higher in the women with the twin gestation (7.3 +/- 0.7 versus 4.7 +/- 0.3 microgram/ml). Although the glucose disappearance rates (K) were not different, there was a significantly lower fasting as well as 5- and 15-minute blood glucose value in the twin pregnancy group. There was also a significantly lower 15-minute insulin level in the twin group. The importance of these findings to the clinical management of twin pregnancies and to the understanding of the metabolic changes in pregnancy is discussed.

Comparison of the fetogram and L/S ratio for fetal maturity.

There were 254 cases studied in which two tests of fetal maturity were performed within 72 hours of each other. The two tests were an x-ray fetogram and an amniotic fluid L/S ratio determination. All fetograms were read by one author for the presence of distal femoral epiphysis (DFE). It was assumed that DFE permits estimation of fetal bone maturation and L/S ratio permits estimation of fetal lung maturity. The DFE results gave a 40% false-positive rate in predicting a mature L/S ratio (greater than 2.0) and a 38.7% false-negative rate. Of the 21 patients who delivered within 3 days of the performance of the two tests, all neonates did well when there were no visible DFE's but a mature L/S ratio. There is a significant lack of specificity and sensitivity of the fetogram DFE's in predicting a mature L/S ratio. Since infant lung maturation is a critical factor in predicting neonatal survival, these results suggest that the x-ray DFE is unacceptable as a single maturity test for use in a perinatal center managing complicated obstetric cases.

Insulin and glucose studies after one year of treatment with a sequential-type oral contraceptive.

PIP: Plasma insulin and blood glucose were measured during an intravenous glucose tolerance test at the University of Minnesota hospital clinic on 28 women on oral sequential contraceptives (15 tablets of 80 mu-g mestranol, 5 tablets of 80 MCG mestranol and 2 mg chlormadinone acetate) both prior to drug initiation and after 1 year of use. No statistical difference in either glucose or insulin levels or weight change was seen after 1 year of therapy. A significant positive association was found between the elevation of glucose and insulin with the drug at cerain time periods during the glucose test, and if there was a family history of diabetes mellitus. A higher frequency of abnormal glucose and insulin levels has been found among users of combination-type drugs as compared to the sequential type.^ieng

Human placental lactogen and intrauterine growth retardation.

Serum human placental lactogen levels were measured after 36 weeks' gestation in 264 serum samples from 109 women with normal pregnancies and in 137 serum samples from 70 women with pregnancies complicated by fetal intrauterine growth retardation (IGR). The fetal and placental weights were significantly lower in the IGR groups while the maternal ages were not different. There was a significantly lower hPL value at each week from 36 to 41 (except for the 39th) in the IGR group. Sixty percent of the women with IGR had hPL values less than 6 mug/ml, and 18.6% were less than 4 mug/ml. It is suggested that a low serum hPL value obtained during the last month of pregnancy should alert the physician to the possibility of intrauterine problems, including IGR.

Effects of norethindrone on carbohydrate and lipid metabolism.

A prospective study was undertaken to investigate the carbohydrate and lipid metabolic effects of the oral contraceptive norethindrone. An oral glucose tolerance test was performed before and 1 year after the daily oral administration of 0.35 mg of norethindrome to 31 women. Measurements were made of glucose, insulin, cholesterol, and triglycerides. During the year there was no significant weight change in the women, and the fasting cholesterol values were unaffected by the steroid. The fasting triglycerides decreased as normally occurs in the late postpartum period. There was no change in the blood glucose curve, but there was a statistically significant elevation produced in all of the plasma insulin values. These data suggest that this 19-nor progestogen steroid can affect the peripheral activity of insulin and thus require higher blood levels in order to obtain the same glucose homeostasis.