Synthetic serum substitute (SSS): a globulin-enriched protein supplement for human embryo culture.

OBJECTIVE: The purpose of the present study was to evaluate whether an IVF protein supplement prepared from human serum albumin (HSA) and human globulins would retain performance characteristics equivalent to those reported for the commercial plasma expanders, Plasmatein (Alpha Therapeutics, Los Angeles, California) and Plasmanate (Cutter Biological, Miles Inc., Elkhart, Indiana). METHODS: Pronuclear-stage human embryos were randomly divided and cultured in human tubal fluid medium (HTF) supplemented with either HSA (5 mg/mL) or Plasmatein (10%, v/v; 5 mg/ml) as a means of indirectly assessing the effect alpha- and beta-globulins have on embryonic development. Those results coupled with the known composition characteristics of Plasmatein were used as the starting basis to formulate test lots of synthetic serum substitute (SSS). RESULTS: Significantly (P < 0.05) more of the human embryos cultured in Plasmatein supplemented medium reached the four-cell or greater stage by 40 hr postinsemination than a comparable group cultured in HSA alone. Lot 1 of SSS, formulated with HSA (84% of total protein) and human globulins (16% of total protein) and an aqueous lipoprotein fraction derived from human plasma (Excyte IV; Miles Diagnostics, Kankakee, Illinois), produced accelerated early embryonic growth relative to control murine embryos grown in the presence of Plasmatein, however, the percentage of the embryos reaching the hatched blastocyst stage was decreased (45 vs 100%). Human embryos from seven patients, randomized to HTF medium supplemented with Plasmatein or lot 1 of SSS, showed equivalent growth at 36-40 hr postinsemination. A microprecipitate developed in media supplemented with lot 1 after several days of culture. The Excyte IV concentration was reduced and, ultimately, eliminated from the subsequent and final prototype lots of SSS. Murine embryos grown in the presence of lipoprotein free SSS showed significantly accelerated (P < 0.01) growth at 17 hr postthaw compared to Plasmatein and all embryos progressed to hatching by 41 hr. Human embryos, randomized to either Plasmatein or lot 3 of SSS, showed significantly accelerated growth (P < 0.01) when scored at 38 hr following insemination. CONCLUSION: Synthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.

Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions.

OBJECTIVE: To determine the effect of medium with or without glucose and phosphate on the fertilization and development of human oocytes. DESIGN: Sequential allocation of alternate patients to one of two treatment groups. SETTING: Private practice infertility programs. PATIENTS: Ten couples requesting treatment for infertility. INTERVENTIONS: Gametes from each couple were collected, washed, and incubated in one of two culture media under investigation. MAIN OUTCOME MEASURES: Number of oocytes collected, fertilized, cleaving, replaced, and implanting in each patient. Development of any supernumerary embryos to fully expanded blastocysts in vitro. RESULTS: There was a significant increase in the proportion of transferred embryos implanting in the group of patients whose gametes were handled in medium devoid of glucose and phosphate. All other comparisons of factors that may have influenced implantation rates between the two groups of patients were not significantly different. CONCLUSIONS: High rates of fertilization, cleavage, implantation, and development of supernumerary human embryos to the blastocyst stage in vitro were obtained with a modified human tubal fluid medium containing ethylenediaminetetraacetic acid and glutamine but devoid of glucose and phosphate ions. A prospective randomized trial is necessary to evaluate the clinical significance of these observations.

A simple method for preparing modified TES and Tris yolk buffer that is optically clear and membrane filterable.

OBJECTIVES: To determine whether modified TES and Tris (TEST) yolk buffer (TYB) made using a commercially available egg yolk extract would exhibit lab performance characteristics equal to the existing preparation made with whole egg yolk and to define the phospholipid content of the new modified TYB formulation. DESIGN: Divided ejaculates from 21 normozoospermic and 7 oligozoospermic males presenting for pre-IVF evaluation were stored at 0 to 4 degrees C for 42 hours using commercially available or modified TYB before analysis in the optimized sperm penetration assay (SPA). SETTING: A commercial tissue culture manufacturer and a clinical fertility reference laboratory. MAIN OUTCOME MEASURES: Sperm swim-up recoveries and average penetrations per ovum, determined by the SPA, were used as measures of sperm function. High-pressure liquid chromatography (HPLC) was used to profile the egg yolk extract. RESULTS: No significant differences in either sperm swim-up recovery rates or SPA results were found in normal or poor quality semen that was treated with modified or commercial TYB. The major constituent in commercial egg yolk extract is lecithin. CONCLUSIONS: Commercially available egg yolk extract passes easily through a 0.2-microns filter, is a rich source of lecithin, and can be substituted effectively for whole egg yolk in preparing TYB.

A new long shelf life formulation of modified Ham's F-10 medium: biochemical and clinical evaluation.

PURPOSE: To evaluate biochemically and clinically a new formulation of modified Ham's F-10 medium made without the inclusion of hypoxanthine. The medium was formulated for long-term storage and use by separately preparing a stable liquid ("basal") portion and a freeze-dried "supplement" containing the labile medium components. RESULTS: Following 18 months of storage the basal medium was biochemically analyzed for its amino acid (aa's) and vitamin content. Cysteine and tryptophan were decreased to less than 30% of their starting theoretical concentrations (STCs). Asparagine, serine, tyrosine, histidine and lysine were present at 50% to 70% of their STC. The remaining aa's were all within 90% of their STCs except arginine which was at 77%. All of the vitamins were present at 90% or more of their STCs except inositol, riboflavin and thiamine which were present at 70% of their STCs. IVF with the new formulation resulted in 13 deliveries from 51 aspirations (25%) as compared with 10/39 (26%) in 1991, when standard medium preparation was used. Oocyte donation resulted in 30 deliveries from 84 cycles (36%) with the new formulation as compared with 21/65 (32%) in 1991. CONCLUSIONS: (1) The new basal with lyophilized supplement formulation produces similar clinical results in the IVF laboratory as medium prepared in the standard fashion, (2) certain amino acids and vitamins are not stable in the liquid basal medium, and (3) the separate formulation of a liquid basal medium with lyophilized supplement is convenient, viable alternative to modified Ham's F-10 medium prepared in the standard manner (i.e., from powder) and may decrease the need for frequent medium preparation.

Comparaçäo entre o teste do hamster e a análise espermática comun numa populaçäo masculina infértil / Comparison betweem the hamster test and commom sperm analysis in the infertile male population

Acredita-se que o teste do hamster (TH) possui uma alta sensibilidade e especificidade, na seleçäo da populaçäo masculina infértil. Uma análise crítica foi executada entre o TH e os parâmetros da análise espermática comum (AEC), com respeito a sensibilidade, especificidade e valores preditivos normais e anormais, durante o seguimento de uma populaçäo infértil. O estudo prospectivo inclui 142 casais inférteis, com duraçäo média da infertilidade entre 5 + 2, 48 ano, e idade dos homens entre 28 e 46 anos. A propedêutica de investigaçäo do fator feminino foi completa, sendo todas as anormalidades presentes corrigidas, antes da inclusäo das pacientes no estudo. Em todos os casos, a permeabilidade tubária foi no mínimo unilateral, e os casais foram acompanhados pelo período de um ano. O TH foi executado segundo Rogers (1979), tendo como principais modificaçöes o emprego do tempo de incubaçäo curto (3-4 horas) e do meio de cultura Ham-F10. O TH mostrou uma baixa sensibilidade (25,8%) semelhante `a aquela da AEC (concentraçäo = 14,5%, motilidade = 15,3% e morfologia = 32,2%). A especificidade do TH foi de 88,8%; todavia, näo diferiu daquela da AEC (concentraçäo = 94,4%, motilidade = 83,3%). O valor preditivo de gravidez com o TH normal foi de 14,8%, comparável com os obtidos com a AEC (concentraçäo = 13,8%, motilidade = 12,5%, morfologia = 15,1%). O valor preditivo da ausência de gravidez através do TH anormal foi de 94,1%; contudo esses altos índices de acerto podem também ser obtidos com a AEC (concentraçäo = 94,7%, motilidade = 86,3%, morfologia = 93%). Dessa forma, pode-se sugerir que, no caso de valores anormais de TH e AEC, o prognóstico de futura gravidez é baixo. Por outro lado, a AEC ainda é o método inicial na avaliaçäo do homem infértil, reservando-se o TH para os casos em que, apesar de AEC estar persistentemente normal, ainda näo surgiu a gravidez

Ultrasound: a technique useful in determining the side of ovulation.

It is often assumed that ovulation commonly occurs from alternating sides each month, but it is unknown whether it can occur repeatedly from the same side. In an effort to answer this question, we selected 39 women from our infertility clinic who had six consecutive courses of ultrasound (US) to monitor ovulation and in whom the side of ovulation was recorded. Nine patients (23.1%) ovulated from the same side for six consecutive cycles, whereas no patient alternated side of ovulation in the same period of time. Stimulation with clomiphene citrate and/or human menopausal gonadotropin that results in single ovulation did not affect this pattern. The data were analyzed statistically with the use of a Markov Chain Model and the chi-square goodness-of-fit; the occurrence of persistent same-side ovulation was significant to P less than or equal to 0.001.

Effect of oral contraceptives on leukocyte phagocytic activity and plasma levels of prostaglandin E2 and thromboxane B2 in normal menstruating women.

Mononuclear and polymorphonuclear cells were isolated from the peripheral blood of normal menstruating women. Four of the subjects were not using oral contraceptives and five were taking various formulations. The women were tested once a week for 12 consecutive weeks. Plasma levels of 6-keto-prostaglandin F2 alpha (6-KF), prostaglandin E2 (PGE2), thromboxane B2 (TxB2), estrogen, and progesterone were measured by specific radioimmunoassays. The phagocytic activity of the mononuclear and polymorphonuclear cells isolated from the peripheral blood was measured with a bacterial phagocytosis and killing assay. The phagocytic activity of both types of cells was depressed perimenstrually in both groups of women. However, examination of individuals showed that those subjects not taking oral contraceptives had a worsening of phagocytic activities with approaching menses while the oral contraceptive subjects generally had an improving of these activities at this time. We were unable to correlate the phagocytic activities with either hormone or prostaglandin levels in the plasma of these subjects. However, the subjects on oral contraceptives had significantly lower levels of PGE2 and TxB2 than those women who were not using oral contraceptives.

A periodic dosing model of fetal alcohol syndrome in the pig-tailed macaque (Macaca nemestrina).

A nonhuman primate on a periodic ethanol dosing schedule should provide a model of fetal alcohol syndrome (FAS) most relevant to the majority of pregnant women who are "social drinkers" and can exercise reasonable control over their ethanol intake. In this pilot study, four pregnant pig-tailed macaques (Macaca nemestrina) received ethanol once a week from 40 days' gestation. Doses were 2.5 gm/kg for three moderate-dose animals (MDAs) and 4.1 gm/kg for one high-dose animal (HDA). Peak blood ethanol levels reached a mean of 240-256 mg/dl for the MDAs and averaged 379 mg/dl for the HDA. Peak acetaldehyde did not vary with dose. One MDA aborted after the first dose. The other three pregnancies were compared with eight to ten control pregnancies, and the infants' development over the first six months was compared with that of the control offspring. Nutritional status of the pregnant females was normal. The fetal heart rate response to maternal restraint was absent in the HDA. Gestational duration and simian Apgar scores were normal. All three infants were abnormally large, and two were also abnormally heavy, with body weight appropriate to skeletal size. Skeletal maturation, judged by ossification and tooth eruption, was not accelerated. The high-dose infant (HDI) was scaphocephalic, with an underdeveloped cranial base and midface, and its brain was small and dysplastic; its reflex, motor, and cognitive development were retarded. One moderate-dose infant (MDI) had some brain abnormalities; it was hyperkinetic and showed developmental retardation on several behavioral measures. The other MDI was normal. We conclude that the periodic model offers an effective means of investigating FAS in M. nemestrina. Furthermore, when nutrition is maintained, intermittent intake of ethanol by the pregnant primate does not necessarily reatard fetal growth.

Early reproductive loss and the factors that may influence its occurrence.

PIP: The elements and magnitude of human pregnancy wastage are summarized with focus on some of the factors that may influence or increase it. Initial attention is directed to consideration of what is known regarding the single largest category of wastage--abortions occurring within the first 12-14 weeks. Cytogenetically, some 61% of these would be abnormal in chromosomal count; structural genetic defects would account for but 3.8% of the observable cases. Of the numerical defects, about 15% would be representative of monosomy, 52% of trisomy, and 20% of triploidy. If the use of endocrine measurements are applied during this time frame, somewhat less than 75% of women who will abort have circulating human chorionic gonadotropin (HCG) levels 1 standard deviation below the normal mean. Possibly the least understood and, consequently, the most underestimated element in pregnancy loss is the contribution of the male partner. During the some 75 days necessary for development and maturation of the human spermatozoa occur numerous steps, some of which are more susceptible than others to fertility-affecting alteration. Among the array of possibilities, genetic defects resulting from either congenital or biologic factors must be considered as a primary grouping. Epididymal transit, with its accompanying maturation of the sperm cell, represents the next major element susceptible to fertility-affecting change. In the female, disruption of the proper functioning of the hypothalamic-pituitary-ovarian axis would contribute to increasing the percentages of reproductive loss. The result of gamete aging would also be an increase in early embryonic and fetal loss.^ieng

Nicotine and its influence on the female reproductive system.

PIP: This brief review focuses on women and how their smoking can influence physiologic events from ovulation to birth, specifically the effects of nicotine, since effects of carbon monoxide in pregnancy have been reviewed extensively elsewhere. The nicotine alkaloid, which enters the body as a gas, has easy access to the brain stem and the central nervous system of the smoker, and it exerts effects on the nervous systems by activating nicotine cholinergic receptor sites. This interaction with the nervous systems has the consequence of altering adenohypophysial, neurohypophysial, adrenal, and catecholamine secretion patterns. These endocrine responses affect the continuum of events from ovulation to birth in the following ways: 1) alteration of H-P-O axis activity affecting ovulation or early tropic support preceding placental function; 2) alterations in oviductal or uterotubal junction activity leading to inappropriate entry of the blastocyst into the uterus; 3) alteration of catecholamine mediated changes in uterine or placental blood flow patterns; 4) fetal hypoxic episodes occurring in response to HbCO accumulation; and 5) inappropriate changes in fetal endocrine function.^ieng

Effect of dexamethasone and acepromazine on plasma androstenedione levels before and after ejaculation of dairy bulls.

Acepromazine administered i.v. to 3 bulls 15 min before semen collection blocked seminal emission and coitus-induced androstenedione release. Dexamethasone or saline treatment had no noticeable effect.

Alcohol, caffeine, and nicotine as factors in pregnancy.

Use of alcohol, caffeine, and nicotine during pregnancy can have serious consequences for the fetus, such as low birth weight, congenital anomalies, or even death. Recent epidemiologic and animal studies indicate that these drugs may exert their effect by a number of metabolic and endocrine mechanisms, which are described here.

Caffeine and pregnancy. A retrospective survey.

A population group consisting of 800 households of women who recently had been pregnant was surveyed to determine the level of consumption of a variety of beverages. Three fourths of the subjects were Mormon. Of a subgroup of 16 women identified as having an estimated daily intake of caffeine of 600 mg or greater, 13 had experienced a reproductive loss in the form of spontaneous abortion (eight) or stillbirth (five), two had given birth to premature infants, and only one had had an uncomplicated delivery. An inordinately high rate or reproductive loss also was noted in 13 households where the man's estimated daily intake of caffeine was greater than 600 mg. A cause-and-effect relationship cannot be determined by this type of retrospective study, but physicians should keep in mind the possibility that an excessive intake of caffeine may be a factor in otherwise unexplainable spontaneous abortion or perinatal mortality.

Caffeine: its direct and indirect influence on reproduction.

Caffeine constitutes the active drug principle in a number of commonly consumed beverages. Among North American adults, it holds the distinction as being probably the most widely utilized psychotropic drug. The compound is most often consumed in the form of coffee, which is derived from the bean of the tropical tree Coffea arabica or Coffea robusta. The unique pharmacologic and physiologic properties of caffeine have led to extensive research efforts, especially in the area of mutagenically and teratogenically mediated effects. In this regard, investigators have often overlooked the alterations in catecholamine, free fatty acid and cyclic nucleotide levels that the the presence of caffeine brings about. Such alterations are discussed in light of their effect on reproduction, and an attempt has been made to clarify the effects that the associated habits of alcohol and smoking have on the reproductive system.

Selected beverage consumption patterns among Mormon and non-Mormon populations from the same geographic location.

This study presents data from a mail questionnaire survey designed to determine the daily beverage consumption patterns of Mormon and non-Mormon households in Utah and southern Idaho. Mormons differed significantly from their non-Mormon counterparts in both their low intake of coffee, tea, and beer, and increased daily consumption of milk and water. A high percentage of Mormons adhere to Church doctrin in their beverage intake habits and the results would appear to substantiate previous preliminary studies of the dietary habits in this religious group.

Whole blood leukocyte culture technique for mammalian cytogenetic analysis.

In the past, the in vitro culture of leukocytes for cytogenetic analysis has required a slightly altered technique for each animal species. This study showed that a simple, yet standardized, in vitro culture system was readily applicable to growth of bovine, canine, equine, feline, ovine and porcine blood leukocytes. The major features of the standardized technique included: 1) the use of whole heparinized blood; 2) a minimum, unfortified culture medium; and 3) a vortex mixer for mixing materials during the hypotonic and fixative stages of the harvesting procedure.

Serum testosterone and estrogen concentrations in the Holstein-Friesian bull after successive ejaculations.

The testosterone and estrogen concentrations in venous serum of 4 mature Holstein-Friesian bulls were determined by radioimmunoassay before and after each of 3 consecutive semen collections taken at 1-hour intervals. The hormonal concentrations were highly variable for the 2 steroids, but uniform in their patterns after the bulls had ejaculated. After the 3rd semen collection, marked changes in hormonal concentrations did not occur.

Caffeine-mediated changes of sex ratio in Chinese hamsters, Cricetulus griseus.

Sixty 6- to 10-week-old male Chinese hamsters were assigned to two groups. In one, the animals were untreated and in the other, they were treated with 0.02 g caffeine/100 ml water orally for 60 days. The sex ratio of the resulting litters showed a significant (P smaller than 0.025) skewing towards females (61.4%) when compared to that of the controls (49.2%).