Clinical implications of amniotic fluid alpha-fetoprotein in twin pregnancy.

Amniotic fluid alpha-fetoprotein (AF-AFP) was measured in 68 pairs of normal twins. Acetylcholinesterase (ACHE) was measured in 23 of those pairs. A significant difference in AF-AFP levels was found in 9/49 (19.4%) of twin pairs of the same sex and 9/19 (47.3%) of twin pairs of opposite sexes (P = .015). Differences in fetal size or gestational age at amniocentesis did not influence the rate of discordant AFP results. ACHE activity was identical in all amniotic fluid samples in twin pairs. In five additional patients, fetal anomalies (three), fetal death or fetal blood in the sample could have affected AFP levels, which were found to be discordant in three of them. Using fetal sex to represent zygosity, the study indicated that discordance in AF-AFP is more common in dizygous twins. The odds ratio for having a discordant AFP result if twins are of different sexes was 4.0. Implications of our data are: (1) in same-sex twins, discordance between sacs is less common, even if only one fetus is affected; (2) the difference in AFP results in twins of the same sex and of different sexes suggests variability in AFP transfer between twin gestational sacs; and (3) ACHE readily diffuses between sacs and cannot be used to determine which twin has an abnormality.

A normal ultrasound does not obviate the need for amniocentesis in patients with elevated serum alpha-fetoprotein.

Elevated levels of maternal serum alpha-fetoprotein (MSAFP) will identify a population at increased risk for specific congenital malformations, which are accurately diagnosed by amniotic fluid AFP and acetylcholinesterase. The risk for spontaneous abortion related to amniocentesis, combined with increasing confidence in the accuracy of ultrasound diagnosis, has caused us to question the need for amniocentesis in the diagnostic workup of pregnancies complicated by elevated levels of AFP in maternal serum. A retrospective study of 257 pregnancies evaluated for elevated serum AFP levels revealed 16 fetal malformations diagnosed by amniotic fluid AFP and acetylcholinesterase. Only 12 of these malformations were diagnosed on the initial ultrasound study. All malformations were diagnosed when ultrasound examination was repeated for increased acetylcholinesterase activity. Earlier gestational age at scanning, smaller defects, and pure technical failure were implicated as causes of misdiagnosis. The rate of fetal malformations identified in this high-risk population (6.23%) and the rate of ultrasound misdiagnosis (1.5% of the population with elevated levels of MSAFP) imply that amniocentesis should still be considered an essential part of the diagnostic workup in these situations.

Amniotic fluid acetylcholinesterase: implications of an inconclusive result.

The implications of an "inconclusive" acetylcholinesterase test (a faint but true band) in amniotic fluid were studied over a 2 1/2-year period in our laboratory. One thousand one hundred fifty-four amniotic fluid samples were tested for acetylcholinesterase and alpha-fetoprotein; the rate of an inconclusive acetylcholinesterase result was 3.3% (38 of 1154). Fourteen such results were found in patients with a high amniotic fluid alpha-fetoprotein level (23.3%), and 24 results were associated with normal amniotic fluid alpha-fetoprotein levels (2.19%). The rates of congenital fetal malformation associated with an inconclusive acetylcholinesterase result in the two groups were 57.14% and 37.5%, respectively. In amniotic fluid samples obtained before 15 weeks' gestation, there was a higher rate of inconclusive acetylcholinesterase tests (9.29%), but a lower percentage of malformed fetuses were found compared with later in pregnancy (2.46% and 56%, respectively). Thus we suggest the terminology "equivocal" for early specimens and "suspicious" for later specimens. If obtained in early second trimester and the ultrasound scan is normal, such findings implicate the need for a careful search for fetal malformations. A positive pregnancy outcome may be expected in most cases.

Amniotic fluid alpha-fetoprotein and acetylcholinesterase in early genetic amniocentesis.

Early identification of fetal abnormalities is possible as a result of improved ultrasound resolution, chorionic villus sampling, and early genetic amniocentesis. A potential advantage of early genetic amniocentesis over chorionic villus sampling is its ability to detect neural tube defects. We obtained 476 amniotic fluid samples between ten and 15 weeks' gestation and analyzed them for karyotype and alpha-fetoprotein (AFP); 142 were also tested for acetylcholinesterase. Amniotic fluid AFP levels rose to a peak at 12-13 weeks' gestation and then gradually declined, closely approximating the pattern in fetal blood. The rate of inconclusive acetylcholinesterase results (a faint but true band) was four times higher than that later in pregnancy (10.6 versus 2.46%, respectively). However, the rate of associated fetal congenital anomalies was lower than that later in pregnancy. Chromosomal abnormalities were detected in the study group, and the association with low amniotic fluid AFP in early genetic amniocentesis levels was identical to that further along in pregnancy. These data help establish normal values for AFP in early pregnancy. With AFP and cautious interpretation of acetylcholinesterase, early genetic amniocentesis can be used for neural tube defect detection.

The use of clinical, biochemical, and ultrasound parameters for the diagnosis of intrauterine growth retardation.

Fifty-four pregnant patients referred for nonstress testing with findings suggestive of intrauterine growth retardation (IUGR) were followed with serial biochemical determinations and ultrasound evaluations. Confirmation of IUGR was made in 18 of the neonates based on body weights below the 10th percentile. Stepwise discriminate function analysis was used to determine the factors most predictive of IUGR among several clinical, biochemical, and ultrasound parameters as well as their combinations. Only the determinations closest to the time of delivery were used in analysis. The presence of abnormal fetal ultrasound measurements had the highest predictive value. Prepregnancy weight improved the prediction slightly with a proportion of correct predictions increasing from 70 to 74%. Only the extremes of prepregnancy weight altered the prediction made on the basis of ultrasound. In contrast, no significant predictive value was demonstrated for weight gain, heavy cigarette smoking, hypertension, serum estriol, human placental lactogen, alpha-fetoprotein, or a decrease in amniotic fluid volume, either singly or in combination with other variables.

Establishment of a collaborative university-commercial maternal serum alpha-fetoprotein screening program: a model for tertiary center outreach.

Expansion of the availability of tertiary level services beyond major medical centers has proved to be a major problem in health care delivery. Routine maternal serum alpha-fetoprotein screening for neural tube defects, and now also for aneuploidy, is a classic example in which there has been a schism between the clinical expertise to manage such a program within a tertiary level reproductive genetics center and the ability to reach patients in regions that are not routinely accessible to the tertiary center. To address this problem we have established a collaborative university-commercial laboratory statewide maternal serum alpha-fetoprotein program that we believe can serve as a model for others. In the first 4 months since its implementation, the program volume has increased tenfold. The detection frequency of neural tube defects has been consistent with that of other programs (1/1690). Three aneuploid karyotypes were found in amniotic fluid of 118 women less than 30 years old who underwent genetic amniocentesis because of a low maternal serum alpha-fetoprotein value. Thus we conclude that: the establishment of a joint university-commercial maternal serum alpha-fetoprotein program may provide a successful model for efficient tertiary center outreach, assessment of our data suggests that a population at high risk for abnormal fetuses can be identified among patients not generally considered at high risk, low maternal serum alpha-fetoprotein values may likely be a more important public health measure than high ones.

Chorionic villi sampling changes maternal serum alpha-fetoprotein.

Second trimester amniocentesis has traditionally been utilized for prenatal genetic diagnosis. Chorionic villi sampling (CVS) is presently offered as an alternative. The occurrence of fetomaternal bleed (FMB) during CVS could increase the rate of post sampling abortion and, additionally, be of significance in patients at risk for isoimmunization. Detection and quantitation of FMB can be accomplished by the determination of changes in maternal serum alpha-fetoprotein (MSAFP) before and after CVS.

Maternal serum alpha-fetoprotein (MSAFP) and fetal growth.

Early mid-trimester screening of maternal serum alpha-fetoprotein (MSAFP) for the detection of neural tube defects is becoming a routine part of obstetrical care. In singleton pregnancies in the absence of fetal chromosomal abnormalities and anatomical anomalies high levels of AFP have been variably related to increased risk for low birthweight infant outcome. The overall relationship, if any, of maternal serum AFP to infant birthweight has, however, not been previously characterized. Between 15 and 20 weeks gestation, MSAFP values were determined for 110 women carrying single, anatomically and karyotypically normal fetuses. Statistical analysis utilizing polynomial and multilinear regression was used to determine the relationship of early mid-trimester MSAFP first to neonatal birthweight and then to gestational age and birthweight adjusted for gestational age. For every increase of one multiple of the median in MSAFP, neonatal birthweight fell 322 grams. This was accounted for almost entirely by decreased fetal growth; early mid-trimester MSAFP was linearly related to birthweight adjusted for gestational age ten times more strongly than to gestational age alone. The explanation for this relationship remains speculative, but the utility of routine AFP screening for the antenatal detection of intrauterine growth retardation certainly deserves further study.

Study on the presence of abnormal proteins in the serum of oral contraceptive users.

Beaumont reported that current and former oral contraceptive users and subjects with thromboembolic disease characteristically demonstrate elevated amounts of precipitated proteins, designated as "globulines anormalement precipitables (GAP)," following treatment of their sera with 25% saturated ammonium sulfate. In the present study, the reproducibility of Beaumont's methods and results was investigated in groups of women consisting of never users, current users, former users, and thrombosis subjects. The current users had the lowest serum GAP levels and no bimodal distribution. Values were higher for the never users and former users and highest in the thrombosis group. Race affected serum GAP values; mean values for blacks were significantly higher than for Caucasians. This study does not support Beaumont's earlier findings.

PIP: 5 groups of never, former, or current oral contraceptive (OC) users or thrombosis patients were subjects of a study of the reproducibility of methods and results published earlier by Beaumont, who reported that current and former OC users and patients with thromboembolic disease characteristically demonstrate elevated amounts of precipitated proteins, designated as "globulines anormalement precipitables (GAP)," following treatment of their sera with 25% saturated ammonium sulfate. Group 1 included 80 women who had never taken OCs; group 2, 17 current users of OCs containing 50 mcg or more of ethinyl estradiol (EE); group 3, 50 current users of OCs containing less than 50 mcg of EE; group 4, 55 women who had taken OCs for at least 2 years but stopped at least 2 months prior to sampling; and group 5, 17 patients with thrombotic and related diseases. Beaumont's procedure for determination of abnormal globulins in serum was followed as closely as possible. The results showed differences in the serum GAP values among the 5 groups, with highest mean GAP value in the thrombosis group, lower in the never users and former users, and lowest in the current users, where estrogen dosage had no effect. The differences were significant only for the 1st sample, not for the average of the 1st sample and a smaller repeat sample taken 1 month later. Mean serum GAP values for blacks were significantly higher than for whites. The pattern of GAP values for the control and experimental groups was different from that obtained in Beaumont's study, in which current and former users exhibited mean serum GAP values that were significantly higher than those of never users. Beaumont's finding of a bimodal distribution of serum GAP values in current users was not repeated. The possible role of the OC formulation in accounting for the discrepancy in results could not be evaluated because the formulations used in Beaumont's subjects were not reported.

A study of sperm acrosin in patients with unexplained infertility.

Radioimmunoassay (RIA) and the fluorometric enzyme method (FEM) were used to study sperm acrosin levels of semen obtained from 13 men of known fertility (group I), 14 male partners of unexplained infertile couples (group II), 4 men among unexplained infertile couples who fathered a child during evaluation and follow-up (group III), and 13 oligospermic males (group IV). Using analysis of variance with the Student-Neuman Keuls post hoc comparisons, we found statistically significant differences between acrosin levels of groups I and II, groups I and IV, groups II and III, and groups III and IV (P less than 0.01) for each comparison and P less than 0.05 for entire experiment). Although RIA was found to be superior to the fluorometric technique, there was excellent correlation between the two methods (r = 0.7; P less than 0.001). This study suggests an association between low sperm acrosin levels and infertility.

Active immunization of female rabbits with purified rabbit acrosin and effect on fertility.

Acrosin immunogen was purified from rabbit testes by sequential acid extraction, ammonium sulfate fractionation, cation-exchange, and affinity chromatography. Twelve females received intradermal injections of purified acrosin in Freund's complete adjuvant followed by a booster injection 6 weeks later. A radioimmunoassay for rabbit acrosin was developed and used to monitor the immune response of the recipients. The females were mated at the time when serum titers of acrosin antibodies were maximal. Four of the animals did not become pregnant, and three of these had the highest antibody titers in the total group. The remaining eight rabbits delivered normal litters at term. Of four control females (immunized with bovine serum albumin), one did not become pregnant. The pregnancy rates for the control and acrosin-immunized rabbits were 75% and 67%, respectively. It is concluded that, although active immunization with acrosin had no significant effect on fertility, the antibody titer produced may be a factor.

Cyclic changes of cervical mucus enzymes related to the time of ovulation II. Amino peptidase and esterase.

The enzymes amino peptidase and esterase were identified in human cervical mucus. Their concentration was serially determined during a menstrual cycle in 5 normal ovulatory women and correlated with the time of ovulation as monitored by the basal body temperature and radioimmunoassay of serum luteinizing hormone (LH), progesterone, and estradiol (E2). The activity of both enzymes decreased at midcycle just before the LH surge and began to rise after ovulation. The preovulatory decline in enzyme activity was significant for esterase but not for amino peptidase. The site of production and functional significance of these enzymes are not at present identified.

PIP: Changes in the cervical mucus content of the enzymes amino peptidase and esterase were determined over the menstrual cycle in 5 normal ovulatory women. The time of ovulation was estimated by recording basal body temperature and radioimmunoassay of serum luteinizing hormone (LH), progesterone and estradiol. Immediately prior to the midcycle surge of LH, both enzymes showed decreased activity, with the decrease in esterase activity being statistically significant (p less than .008). After ovulation, both enzymes showed increased activity. It remains to determine whether these enzymes play a role in the interaction between sperm and cervical mucus.

Cyclic changes of cervical mucus enzymes related to the time of ovulation. I. Alkaline phosphatase.

The concentration of alkaline phosphatase in cervical mucus was serially determined during a menstrual cycle in five normal ovulatory women and correlated with the time of ovulation as monitored by the basal body temperature and radioimmunoassay of serum lutenizing hormone (LH), progesterone, and estradiol. The activity of alkaline phosphatase decreased significantly at midcycle just prior to the LH surge and began to rise after ovulation. Self-detection of cervical mucus alkaline phosphatase may provide a practical method of ovulation prediction.

PIP: The concentration of alkaline phosphatase in cervical mucus was determined during a menstrual cycle and related to the time of ovualtion. Blood samples and cervical mucus were obtained from 5 normal ovulatory women every 2 days before cycle Day 10 and after Day 20 and daily in the midcycle (Days 10-20). All cycles were presumed to be ovulatory on the basis of biphasic basal body temperature and luteinizing hormone (LH), estradiol and progesterone assays. Alkaline phosphatase concentration decreased significantly (p = .03) at midcycle just prior to the LH surge and began to rise after ovulation. It is suggested that combined with the perception of mucorrhea, self-detection of cervical mucus alkaline phosphatase may provide an accurate method of ovulation prediction.

Isolation of a factor from normal human semen that accelerates dissolution of abnormally liquefying semen.

A liquefraction factor from normal human seminal plasma was purified by cation and anion exchange chromatography. It is nondialyzable, heat-labile, and has many properties in common with the seminal protease "chymotrypsin-like enzyme." Its lytic effect is most distinctly observed in abnormal semen that is semisolid or that is solid and fails to liquefy for several hours. Seminal plasmas from certain of these samples have and adverse effect on normal sperm motility. Clinically, the purified seminal liquefaction factor is useful in solubilizing abnormal samples for sperm analysis and homologous artificial insemination.

Studies on the mechanism of action of continuous microdose quingestanol acetate.

The contraceptive mechanism of action of quingestanol acetate administered as a minipill was investigated in five healthy, ovulating women. Each woman served as her own control and was studied during a normal menstrual cycle followed by a cycle in which she received quingestanol acetate, 300 mug/day given orally beginning on cycle day 1, for 30 days. Urinary estrone, estradiol, estriol, total estrogens, pregnanediol; serum progesterone, follicle-stimulating hormone, and luteinizing hormone, together with cervical mucus properties (including viscosity, ferning, spinnbarkheit, cell content, pH, and proteins), sperm transport through mucus, vaginal cytology, and basal body temperature were studied serially during the control and study cycles. Endometrial biopsy specimens were obtained at the end of each cycle. The results indicated that all control cycles were ovulatory. In the treated cycles, endometrial morphology was slightly altered. There was also suppression of preovulatory follicle-stimulating hormone and luteinizing hormone peaks, alteration of urinary estrogens, and a decrease in serum progesterone during the luteal phase. Cervical mucus properties and sperm penetration were inhibited to varying degress during the treatment cycle. These findings suggested that at least three different factors, i.e., alteration of ovulation, disturbances of corpus luteum function, and cervical mucus changes causing inhibition of sperm penetration, were involved in the contraceptive mechanism of microdose quingestanol acetate.

Contraceptive mechanism of microdose norethindrone.

PIP: To determine how microdose progestogens exert their contraceptive mechanism, 5 normal 20-40 year old women (each acting as her own control) were studied during a normal menstral cycle followed by a cycle in which each received orrally 350 mcg norethindrone per day beginning on Cycle Day 1 for 30 days. Results indicated that all control cycles were ovulatory. In the treated cycle, endometrial morphology was altered. There was also significant suppression of preovulatory FSH and LH peaks, alteration of urinary estrogens (either increase or decrease), and marked suppression of progesterone production during the luteal phase. Cervical mucus properties and sperm penetration were inhibited during the treatment cycle. These findings suggest that at least 3 different factors were involved in the contraceptive mechanism of microdose norethindrone: 1) alteration of ovulation and progesterone production by the corpus luteum, 2) cervical mucus changes and inhibition of sperm transport, and 3) endometrial changes.^ieng

Purification and properties of a human seminal proteinase.

1. A method is described for the purification of a proteinase, present in human seminal plasma and previously shown to accelerate migration of spermatozoa through cervical mucus in vitro. A 25-fold purification was achieved in three steps, consisting of ammonium sulphate fractionation, chromatography on CM-cellulose and gel filtration. 2. The enzyme displays some properties similar to chymotrypsin: pH optimum 7.5-8.0; substrate preference of casein, haemoglobin and benzoyltyrosine ethyl ester but not benzoylarginine ethyl ester; mol.wt. 33000. However, it is unaffected by 1mm-di-isopropyl phosphofluoridate or 1mm metal cations, and in this respect differs from chymotrypsin. 3. The properties of the enzyme strongly resemble those of the ;chymotrypsin-like' enzyme discovered in seminal plasma by Lundquist et al. (1955). 4. The use of dimethyl-casein permitted the performance of enzyme assays at substrate concentrations five times higher (up to 50mg/ml) than could be achieved with ordinary casein (10mg/ml).