Yeast mutants conferring resistance to toxic effects of cloned human insulin-like growth factor I.

The production of extracellular human insulin-like growth factor I (IGF-I) in yeast is deleterious to the growth of the host organism. Mutants resistant to the toxic effects of IGF-I production were isolated. A subset of these mutants produced levels of IGF-I greater than the parent strain and were due to chromosomal recessive mutations at a single locus, hpx1. The overproduction of IGF-I was independent of the original promoter and vector expression system. The mutant strains also displayed enhanced extracellular production of other heterologous proteins.

Sperm distribution and degradation in the human female reproductive tract.

PIP: In 26 fertile women studied 25-41 hours after coitus the presence of uterine sperm directly correlated with the density of sperm in the cervical mucus and the hormonal phase of the woman's menstrual cycle. All patients in whom uterine sperm were found at hysterectomy had a cervical mucus sperm density of at least 10/hpf when examined 24 hours before. All were probably in the late proliferative phase or within the period of up to 36 hours after ovulation. In all mammals, following ejaculation most spermatozoa remain in the vaginal cavity. They are subjected primarily to the vaginal fluids and phagocytic cells during the process of degradation. This study confirms a previous hypothesis that from 5 days before ovulation through 2 days after ovulation is the optimal pregnancy period.^ieng

Clinical and ultrastructural findings of an androgenizing Krukenberg tumor in pregnancy.

A clinical and ultrastructural study of an androgenizing Krukenberg tumor in pregnancy is presented. Ultrastructural observations suggested the abundant, hyperplastic, luteinized ovarian interstitial cells as the probable cause of elevated circulating levels of testosterone (5400 ng/dL). The unusual fine structure of these cells, which included large intramitochondrial lipid droplets and abundant smooth endoplasmic reticulum, was indistinguishable from that reported to occur in Leydig cells stimulated by exogenous gonadotropins.

A randomized clinical trial comparing Pipelle and Tis-u-trap for endometrial biopsy.

Recent publications have suggested that use of the Pipelle endometrial suction curette is a safe and effective method by which to obtain samples of endometrial tissue. To address this issue, we performed a randomized clinical trial comparing the Pipelle to the Tis-u-trap in 156 patients. The Pipelle was as effective as the Tis-u-trap in obtaining endometrial samples in both the adequacy of the specimen (Pipelle 88%, Tis-u-trap 84%) and the quality of the specimen (P = .26). This trial confirms the favorable observational reports on the use of the Pipelle for endometrial biopsy.

Effect of supplementary oral estrogen on long-acting injectable progestogen contraception.

PIP: To test the effectiveness of daily oral doses of estrogen in regulating the menstrual cycle of women receiving 150 mg of depo-medroxyprogesterone acetate (DMPA), 32 women were administered 1 mg of stilbestrol daily in conjunction with their DMPA injections for 90 days. A control group of 31 women received DMPA with a placebo for the same period of time. After the first 90 days, 28 women were chosen from both the stilbestrol group and placebo group, and were instructed to take 1 mg of stilbestrol daily for 3 weeks of each of the 3 subsequent 4 week periods, with 22 women receiving no medication. Continuous daily doses during the first 90 days failed to diminish the incidence of uterine bleeding of the silbestrol group when compared to the controls. Supplemental administration of estrogen did not significantly alter the appearances of endometrial biopses or kayopyknotic indices. Cyclic administration of oral estrogen for 3 weeks of a 4 week period caused regular withdrawal uterine bleeding. It was concluded that there was very little advantage to the administration of supplemental oral estrogen to women using DMPA, except in the case of a women who psychologically needs a bleeding period.^ieng

Norethindrone acetate and estradiol-induced endometrial hyperplasia.

OBJECTIVE: To identify the lowest effective continuous dose of norethindrone acetate that significantly reduces 12-month incidence of endometrial hyperplasia associated with unopposed 17beta-estradiol (E2), 1 mg. METHODS: In a double-masked, randomized, multicenter study, 1176 healthy postmenopausal women 45 years of age or older without evidence of endometrial abnormalities were given 12 months of treatment with unopposed E2, 1 mg, or continuous-combined regimens of E2, 1 mg, and norethindrone acetate, 0.1 mg, 0.25 mg, or 0.5 mg. Endometrial histology was evaluated at the end of the treatment period. RESULTS: Continuous-combined E2-norethindrone acetate regimens significantly reduced 12-month incidence of endometrial hyperplasia compared with unopposed E2 1 mg (P <.001). Endometrial hyperplasia occurred in 14.6% of women treated with unopposed E2 1 mg, whereas in all continuous-combined groups, the rate decreased to less than 1%. Among patients who received E2-norethindrone acetate 0.1 mg, incidence was 0.8%; among those who received 0.25 mg and 0.5 mg, it was 0.4%. CONCLUSION: Continuous norethindrone acetate at doses as low as 0.1 mg combined with E2 1 mg effectively negated risk for endometrial hyperplasia associated with unopposed E2 1 mg, at least for the first year of therapy.

Effects of intrauterine devices on nuclear deoxyribonucleic acid metabolism in rabbit blastocysts: an autoradiographic study.

A metabolic parameter, DNA synthesis, was measured in rabbit blastocysts exposed to Silastic intrauterine devices (IUDs) and compared with those without IUD exposure. Autoradiographic detection of 3H-thymidine uptake showed that the IUD-exposed blastocysts contained fewer labeled cells than their IUD-free equivalents. The depressed DNA synthesis may be caused by either a block in the cell cycle or a block in DNA synthesis itself. Either way, the result could be consistent with the observation that embryo deaths occur at all growth stages, preimplantation, during implantation, and postimplantation.

Comparison of techniques for selection of motile spermatozoa.

Procedures to separate motile sperm with high rates of recovery may have clinical application in in vitro fertilization and intrauterine insemination in increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. A two-step continuous Percoll gradient was an effective means of separating motile sperm which also had enhanced ability to penetrate zona-free hamster ova. However, the requirement for a high-speed centrifuge and rotor makes the procedure impractical in many cases. A one-step discontinuous Percoll gradient was also effective in separating a population of motile sperm. Comparison of the discontinuous Percoll gradient with other techniques for separation of motile sperm indicated the discontinuous Percoll gradient had advantages in terms of recovery, enhancement of motility, and increased ability to penetrate zona-free hamster ova. The velocity of selected sperm was not significantly different among techniques. The one-step discontinuous Percoll gradient appears to have value both for increasing homogeneity of human sperm populations used for basic research and in clinical practice for male subfertility.

Uterine bleeding in the rhesus monkey in response to various designs of intrauterine devices.

PIP: To study quantitative menstrual blood loss (MBL), 10 normally rhesus monkeys and 18 ovariectomized, hormonally supplemented monkeys (estradiol and progesterone) were inserted with IUDs: a silastic cylinder and 3 modifications the polyethylene Tatum T device. A 92% increase (p.01) in MBL was noted in the Y-T device monkeys (n=7) and a 70% increase (p.05) in the large oval T ones (n=5). MBL was unaffected by the small oval T, and by the cylindrical Silastic devices after the second postinsertion period. Intermenstrual bleeding was observed in 4 of 7 Y-T device animals but was uncorrelated with MBL. It was concluded that rhesus monkeys may serve as good research models for designing human IUDs.^ieng

Prevention of endometrial hyperplasia by progesterone during long-term estradiol replacement: influence of bleeding pattern and secretory changes.

OBJECTIVE: To determine the relative influences of induction of withdrawal bleedings secretory transformation, and reduction of mitosis in glands on prevention of endometrial hyperplasia during long-term hormonal replacement therapy. DESIGN: Observational expanded clinical case report. SETTING: Reproductive Endocrine Department of Hospital Necker, Paris, France, and Pathology Department of Women's Hospital, Los Angeles County and University of Southern California Medical Center, Los Angeles, California. PATIENTS: Postmenopausal women seeking treatment for symptomatic menopause. INTERVENTIONS: Endometrial biopsy and/or ambulatory hysteroscopy. MAIN OUTCOME MEASURE: Endometrial histology including progestational maturation patterns and glandular epithelial mitosis rates. Macroscopic endometrial appearance. RESULTS: The use of larger doses of E2 and P induced more marked secretory changes and more frequent withdrawal bleeding than the lower doses. There was no evidence of endometrial hyperplasia after 5 years of E2/P replacement therapy independently of bleeding pattern or progestational maturation. Consistent reduction of mitosis rates in glandular epithelium was found after 9 or more days of P administration in each cycle. CONCLUSIONS: Control of endometrial growth is mainly related to control of mitosis in glands by a relatively low doses of P. Induction of withdrawal bleeding and endometrial secretory transformation, which require larger doses of Progesterone, do not provide additional benefit for prevention of hyperplasia. Induction of amenorrhea with a relatively low dose of P may be offered to women seeking hormone replacement therapy with similar levels of safety.

Menstrual blood quantitation in the rhesus monkey: an experimental tool for improving intrauterine contraceptive devices (IUDS).

PIP: 6 normally menstruating and 21 ovariectomized rhesus monkeys were used to measure the menstrual blood loss (MBL) during an estrous cycle. A reliable method of quantitating MBL should provide better evaluation of the bleeding problem associated with IUDs. The technic used in measuring the MBL and the volume of MBL are presented. The ovariectomized animals were given 25-mcg estradiol im daily for 22 consecutive days of each month and 1.5-mg progesterone daily during the second half of the injection schedule. These injections supported normal endometrial histology. Intravaginal pads were used for the collection of the menstrual blood. MBL was calculated by the Hallberg-Nillson method. Average MBL for all menstrual periods was 2.01 ml. This should provide a necessary parameter for evaluation of IUD-associated uterine hemorrhage.^ieng

Results of tubal embryo transfer in premature ovarian failure.

The transfer of embryos generated in vitro to the fallopian tubes in 11 cases of premature ovarian failure resulted in 9 clinical pregnancies. This approach may have theoretical advantages over GIFT and IVF-ET in agonadal patients.

Ultrastructural hemostasis in response to vascular injury induced by intrauterine devices in human endometrium.

PIP: 8 micrographs are presented to help elucidate the mechanism of endometrial bleeding caused by IUDs. A previous study, which reported endometrial vessels with defects or gaps in the superficial portion of IUD-exposed endometrium, is expanded. Since the endothelial cells forming these gaps were in stages of degeneration to complete necrosis, perhaps IUD/endometrial interactions injured some of the superficial vessels which then degenerated, formed gaps, and allowed blood to escape. However, the same endothelial degeneration should initiate platelet adhesion, aggregation, and thrombosis. Yet the micrographs showed degenerated endothelial cells at the vessel gaps, gaps which represent disintegrated endothelial cells, and collagen which appeared to be directly exposed to luminal blood through the gaps. All of these hemostasis-initiating conditions resulted in a surprizing low level of activity. Rarity of platelet and/or fibrin thrombi plugging vessel gaps in IUD-exposed endometrium supported the feasibility of bleeding through small gaps. The interstitial hemorrhage is apparent by electron micrograph. When the concentration of red cells within the superficial endometrium became sufficiently high, apparently the erythrocytes either exuded through the spaces between surface epithelial cells or ruptured into the uterine caivty and resulted in clinical bleeding.^ieng

The effects of progesterone and progestins on endometrial proliferation.

A total of 99 premenopausal and 27 postmenopausal women were evaluated to determine the quantity of glandular proliferation resulting from progestin inhibition of estrogen-primed subjects and of subjects without hormonal stimulation. Endometrial glandular proliferation rates were determined by using mitosis counts, proliferating-cell nuclear antigen (PCNA), and nuclear cyclin (MIB1) immunocytological staining. The endometria of normally cycling premenopausal women, of women who received a synthetic progestin, and of untreated postmenopausal women were studied. In untreated normally cycling premenopausal women, the proliferation of the glandular epithelium was increased during the follicular phase and decreased during the luteal phase. Premenopausal women receiving a synthetic progestin and untreated postmenopausal women who were not estrogen-primed showed minimal epithelial proliferation. Endometrial glandular proliferation is inhibited by endogenous progesterone in premenopausal women. Endometrial proliferation is markedly reduced in premenopausal women receiving a synthetic progestin and in untreated postmenopausal women.

PIP: Use of micronized progesterone or a synthetic progestin has been shown to counter the proliferative effect of estrogen on the endometrium in pre- and postmenopausal women. The present study measured endometrial glandular proliferation rates in 99 pre- and 27 postmenopausal US women. Determinations were based on mitosis counts and both proliferating cell nuclear antigen and nuclear cyclin immunocytologic staining of endometrial tissue. In the untreated, normally cycling premenopausal subjects, glandular epithelial proliferation increased during the follicular phase and decreased during the luteal phase. Premenopausal women who received a synthetic progestin and untreated postmenopausal women who were not estrogen-primed showed minimal epithelial proliferation. The mean mitosis rate of proliferative phase glands was 12.3 compared with 1.6 and 0.01 after administration of the oral contraceptives norethindrone or norethynodrel, respectively. Among premenopausal women, the intensity of the stromal pseudodecidualization and inhibition of glandular development was greatest in those receiving monthly medroxyprogesterone acetate injections. The combination of progestin potency, dosage, and duration determined the mitoses, stroma, and glands that were present in the three groups of subjects. The methods used in this study may be of use in determining optimal dosages of exogenous progestins in women who are receiving hormone replacement therapy and the potential exists for predicting adverse endometrial responses to progestational therapy.

Quantitation of menstrual blood loss--further evaluation of the alkaline hematin method.

PIP: An evaluation of the photometric alkaline hematin procedure was carried out to determine its reliability and practicality in measuring menstrual blood loss (MBL). A reliable method for quantitating MBL was necessitated in order to measure the effect of IUD design on uterine bleeding. MBL was measured in 6 women during 2-4 consecutive cycles (total of 15 cycles). Test samples of blood, either as liquid or soaked into sanitary napkins, vaginal tampons, or cotton pads were incubated at room temperature in 5% (w/v) aqueous sodium hydroxide solution. After incubation, absorbance of the brown-colored alkaline hematin was measured at 550 nm in a Perkin-Elmer Model 124 dual beam spectrophotometer against a blank of either 5% sodium hydroxide or distilled water. The quantity of blood in each sample, represented by eluted, hematin-converted hemoglobin, was determined from a standard curve relating various known volumes of incubated blood to absorbance at 550 nm (A550). Standard volumes of blood were always incubated for the same length of time as the samples. When the samples were diluted with more sodium hydroxide solution than the standards, sample absorbances were multiplied by an appropriate dilution factor. The photometric alkaline hematin procedure indicated a sensitivity to less than .1 ml of blood, within-batch precision of 5% or less (2 C.V.), and an accuracy of measuring human menstrual blood from most sanitary devices generally within plus or minus 5%. The method appeared to be specific for menstrual blood and independent of other materials in genital fluids. Menstrual discharge could be stored for at least 1 month prior to determination without alteration of results. The procudre, once optimized for the authors' purposes, was found to be very practical. The results of the study were consistent with the available literature which indicated that maximum normal menstrual blood flow is between 60 and 80 ml, and the mean is near 30 ml. Variation of measured flow between consecutive menstrual cycles in some individuals (both normal and menorrhagic) revealed the necessity for repeated measurements to properly assess average blood flow in certain cases.^ieng

Influences of copper and design of intrauterine devices on endometrial bleeding in monkeys.

PIP: Uterine bleeding in 8 rhesus monkeys was compared in response to plain vs. a specially fitted copper-wrapped IUD. All animals had utero-cutaneous surgical fistulas for accurate IUD insertion and removal. Daily observations for uterine bleeding between menstruations and quantitation of menstrual blood loss (MBL) were made. Following control periods of observation, each monkey was inserted with either a plain or a copper-wrapped IUD and then studied for 4 consecutive menstrual periods. Each IUD was especially fashioned to an estimate of individual uterine cavity size. Some were wrapped with pure copper wire of 30 sp mm surface area. In some animals the devices were later removed from the uterus, the copper wire taken off, and the plain polyethylene oval-Ts reinserted. In some animals originally wearing plain polyethylene devices, the IUDs were removed, wrapped with copper wire, and reinserted into the same monkeys. During the observed 4 consecutive preinsertion periods, MBL averaged 1.54 ml and was the same in the 4 cycles in which a plain polyethylene oval-T device was in place. No intermenstrual bleeding was observed, except slight spotting for the first 1 or 2 days. During the 4 cycles, with the copper-wound device present, mean MBL was 1.28 ml. Having individually fitted each uterine cavity with a best-fitting IUD was considered important. The addition of copper had not increased the bleeding. In humans, especially fitted contraceptive devices may minimize complications of bleeding and pain as compared with standard designs. Bleeding and pain have been the most frequent complications leading to removal of IUDs.^ieng

Intrauterine medication with epsilon aminocaproic acid. Effect on Rhesus monkeys wearing intrauterine devices.

PIP: Results of tests based on the hypothesis that sustained release of an antifibrolytic agent from an IUD may control the bleeding complications associated with IUD contraception are reported. Devices releasing epsilon aminocaproic acid (EACA) were made from silicone rubber encased in nylon-baked millipore tubes. These devices were cylindrical with a diameter of 1.9 mm and lengths of 14 mm. For easy removal a central core of stainless steel extended beyond 1 end to form a closed loop. Menstrual blood loss (MBI) in a group of Rhesus monkeys was quantitated before and after insertion of a nonmedicated silicone device, a standard polyethylene device, or medicated silicone devices. A beneficial effect of medication with EACA was demonstrated. With nonmedicated silicone devices MBL increased 96% over preinsertion values. EACA-releasing devices caused only a 33% increase during the first menstrual period after insertion. Mean MBL was increased 109% in animals wearing polyethylene IUDs alone and 132% in those wearing polyethylene plus control silicone devices. MBL was increased only 44% in monkeys wearing polyethylene plus EACA medicated silicone devices. In animals with EACA devices no local or disseminated intravascular coagulation was detectable by assay of circulating levels of fibrinogen, plasminogen, or fibrin-fibrinogen degradation products. Although the effects of EACA occurred at a high intrauterine dose for only 1 menstrual period, a much smaller dose might be effective. Studies are being made to fabricate a device that would last for 1 year or more in humans. Complete inhibition of fibrinolytic activity in the uterus at the time of menstruation would be harmful so it is not sought.^ieng

Endometrial responses to various hormone replacement regimens in ovarian failure patients preparing for embryo donation.

Various regimens are recommended for replacing sex steroids in ovarian failure patients attempting donor embryo transfer. We histologically assessed endometrial biopsies obtained on simulated cycle day 26 from functionally agonadal patients (n = 19) receiving hormone replacement according to three different regimens: Regimen 1, oral micronized estradiol (E2) 2 mg days 1-5, 4 mg days 6-9, 6 mg days 10-13, 4 mg days 14-28, with progesterone vaginal suppositories, 100 mg day 15 followed by 200 mg days 16-28; Regimen 2, oral micronized E2 1 mg days 1-5, 2 mg days 6-9, 6 mg days 10-13, 2 mg days 14-28, with progesterone vaginal suppositories, 100 mg day 15 followed by 200 mg days 16-28; Regimen 3: oral micronized E2 1 mg days 1-5, 2 mg days 6-9, 6 mg days 10-13, 2 mg days 14-28, progesterone 50 mg intramuscularly delivered day 15 followed by 100 mg intramuscularly days 16-28. Biopsies were interpreted according to Noyes criteria. While all regimens resulted in variable degrees of stromal pseudodecidualization, Regimen 1 biopsies uniformly demonstrated glandular abnormalities consistent with excessive estrogen stimulation. This included aberrant maturation, intraluminal papillary excrescences and variations in epithelium size and stratification. Regimen 2 biopsies were morphologically normal in most patients, yet many manifested minor variations in gland maturity. Only Regimen 3 biopsies were consistently normal on day 26 or slightly advanced in maturation. We conclude that endometrial morphology differs according to the hormone replacement preparation and route of administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Postmenopausal hormone replacement with a combination estrogen-progestin regimen for five days per week.

A group of 15 postmenopausal women who had not recently received estrogen replacement were enrolled in a study during which they received 0.625 mg of conjugated equine estrogen and 2.5 mg of medroxyprogesterone acetate daily from Monday through Friday of each week for six months. No treatment was given on the weekend. Endometrial biopsy specimens at the end of therapy revealed minimal growth of glands and stroma and a low mean mitosis count. Of the 12 women who completed the trial, 5 were completely amenorrheic, and only 4 of the 15 bled beyond the second month of treatment. Of those four, two spotted for only a few days. In the 12 women who completed the trial there was a significant increase in high density lipoprotein cholesterol and a nonsignificant lowering of low density lipoprotein cholesterol. The results of this study indicate that comparative trials between this regimen and one in which the two drugs are given daily for seven days a week are warranted.

Cytodiagnosis of Candida organisms in cervical smears.

Cervical smears and cervical scrapings cultured on Sabouraud agar from 31 women suspected of having Candida genital infections were examined in a study of the cytomorphology of this fungal infection in cervical smears. Of the 31 samples, 20 (64.5%) grew C. albicans in culture. One sample (3.2%) grew C. paratropicalis, 2 (6.4%) grew mixed C. albicans and Torulopsis glabrata and 2 (6.4%) grew T. glabrata alone. Of the 25 fungus-positive samples, 20 (80%) had fungus-positive cervical smears and 5 (20%) had fungus-negative smears. There was no instance in which the cervical smear was positive but the culture was negative. Among the cases positive for C. albicans, organisms occurred in two forms: pseudohyphae without blastospores (29.4%) and pseudohyphae with blastospores (70.6%). T. glabrata was present in the smears as budding and nonbudding yeasts. Although the sensitivity of the cervical smear in detecting fungus in culture-positive patients was only 80%, the cervical smear can still be a useful means of rapid identification of C. albicans when blastospores and pseudomycelium are present. The presence of budding or nonbudding yeast without pseudohyphae should strongly suggest a T. glabrata infection.