In vitro differentiation of bovine bone marrow-derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors.

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFß1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFß1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21-day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFß1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.

Concentrations of Mercury and Other Inorganic Ions in Wet Precipitation Collected from a Mountain Mining Zone and an Urban Area in Central Mexico.

We measured and compared mercury (Hg) and other ions in rainwater collected in San Joaquin (mining zone) and Juriquilla (urban area), central Mexico, from 2009 to 2012. A total of 274 rainwater samples were collected and analyzed for pH, electrical conductivity, [Formula: see text] Cl-, [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg. Mercury concentrations in rainwater varied from 24.21 to 248.89 (x-bar = 86.97 ± 10.77) µg L- 1 in San Joaquin (mining zone) and 11.26 to 176.91 (x-bar = 81.51 ± 10.24) µg L- 1 in Juriquilla (urban area). Rainwater sample were collected over periods 1-3 days, depending upon precipitation frequency. Significant correlations (p < 0.05) were found between [Formula: see text] Cl-, [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg at the San Joaquin site. Significant correlations were obtained between [Formula: see text] Na+, K+, Ca2+, Mg2+ and Hg at the Juriquilla site. In order to determine if there were significant differences among each measured parameter in rainwater collected in San Joaquin and Juriquilla, Kruskal-Wallis test was applied to data. We emphasized that the distribution and concentrations of Hg and the studied ions in rainwater samples were affected by atmospheric dust and local meteorological conditions of wind-speed and direction.

Current situation of polycyclic aromatic hydrocarbons (PAH) in PM2.5 in a receptor site in Mexico City and estimation of carcinogenic PAH by combining non-real-time and real-time measurement techniques.

Air pollution is a public health concern. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous atmospheric pollutants contained in the atmospheric aerosol. PAH in particulate matter with diameters ≤2.5 µm (PM2.5) represent a human health risk due to their toxic properties. In this study, PAH in PM2.5 at a receptor site of Mexico City during the dry cold season were determined. The most abundant PAH (median, 10-90th percentile, pg m-3) were benzo[ghi]perylene (467, 291-697), followed by pyrene (427, 218-642). A decrease around 40% in the carcinogenic PAH onto PM2.5 was calculated with respect to the same PAH measured a decade ago, at the same receptor site, despite of increase in vehicle fleet. The PAH decrease trend agrees with the decrease trend of CO, NO and NO2, released into the air by similar emission sources than PAH. Control emissions strategies implemented by local and federal authorities are discussed. PAH analyses were carried out by non-real-time and real-time methods. The PAH non-real-time method involved PM2.5 sampling, sample treatment and gas chromatography-mass spectrometry analysis. The PAH real-time method involved the use of a photoelectric aerosol sensor (PAS). The PAH determination by non-real time method was selective and efficient, with recoveries between 75 ±â€¯14% and 98 ±â€¯26%. By combining non-real-time and real-time methodologies, multivariate regression models were obtained based on PAS response, NO2 and wind speed to estimate PAH in PM2.5 at low-cost (r2 = 0.59 to r2 = 0.89). Fossil fuel combustion from vehicles was the major source around the sampling site. Diagnostic ratios (DR) based on retene, chrysene, and triphenylene, suggested biomass burning emission sources. Photo-oxidation in sunny months was observed based on benzo[a]pyrene, benzo[ghi]perylene, benz[a]anthracene, indeno[1,2,3-cd]pyrene and black carbon. The correlation analyses suggested transport of PM2.5, O3, BC and SO2 to the sampling site, and local emissions of PAH, NO and CO.

Safety and efficacy of a mesenchymal stem cell intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis.

Although, antibiotics are effective in the treatment of bovine mastitis, they do not address the regeneration of mammary glandular tissue and have been associated to the increment in antimicrobial resistance worldwide. Considering the necessity of alternative therapies for this disease of high economic impact and the reported regenerative and antibacterial effects of mesenchymal stem cell (MSCs), we evaluated the safety and efficacy of an allogenic MSC-based intramammary therapy in dairy cows with experimentally induced Staphylococcus aureus clinical mastitis. In a safety trial, heifers were inoculated intramammarily with a 2.5 × 107-suspension of bovine fetal AT-MSCs on experimental days 1 and 10. Animals were evaluated clinically on a daily basis during a 20-day experimental period and blood samples were collected for hemogram determination and peripheral blood leukocytes (PBLs) isolation. In an efficacy trial, Holstein Friesian cows were inoculated with S. aureus and treated intramammarily with vehicle (NEG; days 4 and 10), antibiotics (ATB; days 4 and 5) or a suspension of 2.5 × 107 AT-MSCs (MSC; days 4 and 5). Cows were clinically evaluated daily and milk samples were collected for somatic cell count (SCC) and colony forming units (CFU). Blood samples were collected for serum haptoglobin and amyloid A determination. Intramammary administration of two doses of bovine fetal AT-MSCs in healthy cows did not induce changes in clinical or hematological variables, and gene expression profiles in PBLs associated to activation (CD4, CD8, CD25, CD62L and CD69) and proinflammatory cytokines (CCL2, CCL5, IL2, CXCL3, IFNγ, and TNFα). Quarters of MSC group of cows had similar SCC log/mL in milk compared to infected quarters of ATB or NEG cows. However, quarters of MSC cows had lower CFU log/mL in milk compared to quarters of NEG cows. Intramammarily inoculation of repeated doses of 2.5 × 107 allogenic AT-MSCs did not induce clinical or immunological response in healthy cows. Moreover, MSC-intramammary treatment reduced bacterial count in milk of cows with S. aureus clinical mastitis compared to untreated cows. This work provides initial evidence for the safety and efficacy of an allogenic MSC-based intramammary therapy for the treatment of bovine mastitis.

Comparative analysis of in vitro proliferative, migratory and pro-angiogenic potentials of bovine fetal mesenchymal stem cells derived from bone marrow and adipose tissue.

Mesenchymal stem cells (MSCs) are found in virtually all tissues, where they self-renew and differentiate into multiple cell types. Cumulative data indicate that MSCs secrete paracrine factors that may play key roles in the treatment of various acute and chronic pathological conditions in diverse animal species including cattle. The aim of the present study was to compare the potentials for proliferation, migration and pro-angiogenesis of bovine fetal BM-MSCs and AT-MSCs under in vitro conditions. Growth curves and population doubling time (PDT) were determined for BM-MSCs and AT-MSCs in order to compare in vitro cell proliferation potentials. The ability of BM-MSCs and AT-MSCs to migrate was evaluated by scratch plate and transwell migration assays. The pro-angiogenic potential of conditioned medium from BM-MSCs and AT-MSCs was compared using an endothelial cell (EC) tubule formation assay. BM-MSCs displayed higher proliferation curves and doubled their populations in fewer days compared to AT-MSCs. No significant differences were detected in the number of migrant cells between BM-MSCs and AT-MSCs; however, a higher migration value was detected for BM-MSCs compared to fibroblasts (FBs), and a higher number of migrant cells were attracted by DMEM supplemented with 5% fetal bovine serum (FBS) compared to stromal cell-derived factor-1 (SDF-1). More tubules of ECs were formed after exposure to concentrated conditioned medium from AT-MSCs compared to BM-MSCs, FBs or DMEM controls. Despite common mesodermal origin, BM-MSCs display higher proliferative capacity and lower pro-angiogenic potential compared to AT-MSCs; however, both cell types possess similar migratory ability.

Sertoli cell-mediated differentiation of bovine fetal mesenchymal stem cells into germ cell lineage using an in vitro co-culture system.

In vitro gamete derivation based on differentiation of germ cells (GC) from stem cells has emerged as a potential new strategy for the treatment of male infertility. This technology also has potential applications in animal reproduction as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to differentiate into mesodermal lineages. Under the effect of selected bioactive factors, MSC upregulate expression of pluripotent and GC specific-markers revealing their potential for GC differentiation. In addition to the effect of trophic factors, cell-to-cell interaction with Sertoli cells (SC) may be required to guide the sequential differentiation of MSC into GC. Thus, the aim of the present study was to investigate the effect of coculture with SC on the potential for in vitro GC differentiation of bovine fetal MSC (bfMSC) derived from bone marrow (BM-MSC) and adipose tissue (AT-MSC). bfMSC were isolated from male bovine fetuses and SC were collected from adult bull testes. The effect of SC interaction with BM-MSC or AT-MSC was analyzed on the expression of pluripotent factors OCT4 and NANOG, GC genes FRAGILLIS, STELLA and VASA and male GC markers DAZL, PIWIL2, STRA8 and SCP3 at Day 14 of coculture. Flow cytometry analyses detected that the majority (95,5% ±â€¯2.5; P < 0.05) of the isolated population of SC cultures were positive for SC-specific marker WT1. Levels of mRNA of WT1 in BM-MSC and AT-MSC were lower (P < 0.05) compared to SC; whereas, WT1 expression was not detected in bovine fetal fibroblasts (FB). Cocultures of BM-MSC and AT-MSC with SC had higher (P < 0.05) OCT4 mRNA levels compared to monocultures of BM-MSC, AT-MSC and SC. Moreover, cocultures of BM-MSC with SC had higher (P < 0.05) proportion of cells positive for Oct4 and Nanog compared to monocultures of BM-MSC and SC. Levels of mRNA of DAZL, PIWIL2 and SCP3 were upregulated in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Accordingly, the proportion of cells positive for Dazl were higher (P < 0.05) in cocultures of AT-MSC with SC compared to monocultures of AT-MSC and SC. Changes in gene expression profiles during coculture of SC with AT-MSC suggest that cell-to-cell interaction or bioactive factors provided by SC may induce progression of AT-MSC into early stages of GC differentiation.

Ram semen deterioration by short-term exposure to high altitude is prevented by improvement of antioxidant status.

Ovine reproduction efficiency in herds at high altitude (ha) is lower than that at low altitude (la). In ewes, ha effects are due to hypoxia and oxidative stress. Our aim was to establish the effect of antioxidant vitamin supplementation on semen traits and antioxidant status of rams exposed to short or long time ha. A total of 32 rams native to la (~500 m) were used, 16 were kept at la and the other 16 were brought to ha (~3600 m), where they were placed in the same flock as the ha native rams (n=16). Half of the animals in each group were supplemented daily with vitamins C 600 mg and E 450 IU per os, during the entire experimental period, starting the 4th day after animal's arrival at ha (day 0). At days 0, 30 and 60 of treatment, blood and semen samples were collected for evaluation of antioxidant status and semen standard characteristics. Data were compared within each experimental time by analysis of variance using a general linear model. Elevated concentrations of oxidative stress biomarkers were present in blood from animals maintained at ha. Ejaculates from ha exposed rams showed decreased sperm concentration, progressive motility and viability, in addition to decreased antioxidant status in seminal fluid. A total of 30 days of oral supplementation with vitamins C and E prevented some ha negative effects on semen characteristics, mainly in recently ha exposed rams. It is concluded that exposure of rams to ha negatively affects semen quality, where oxidative stress plays a predominant role. These effects are mainly prevented by oral supplementation of vitamins C and E, which constitutes a simple and cheap alternative to improve semen quality of rams when they are moved to ha.

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and ß-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and ß-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3ß-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

Comparison of three estrus detection systems during summer in a large commercial dairy herd.

The objective of the study was to compare three systems for estrus detection and combinations of these systems on a large commercial dairy (1075 lactating cows) during stress of summer heat. At 37-45 days in milk (DIM), 255 cows were fitted with a HeatWatch device (HW; DDx Inc., Denver, CO), an activity sensor ALPRO (ALPRO; DeLaval Inc., Kansas City, MO), and visually observed (VO) three times daily. Pregnancy status was determined by uterine palpation per rectum 35-49 days following artificial insemination (AI). Effects of DIM, parity, standing events, inseminator, and interval between onset of estrus and AI on conception rates were determined using logistic regression. Efficiencies for detection of estrus, determined by comparing detected periods of estrus with a theoretical total of 570 periods, were 49.3% (VO), 37.2% (ALPRO), 48.0% (HW), and 80.2% for all three systems simultaneously. Conception rates (LSM+/-S.E.) for cows detected by one or more of the three systems were 6.2+/-3.9 for VO, 19.8+/-5.6 for ALPRO, 17.3+/-5.0 for HW, 22.8+/-7.0 for VO+ALPRO, 26.9+/-4.6 for VO+HW, 23.2+/-5.2 for ALPRO+HW, and 18.4+/-4.7 for VO+ALPRO+HW. Inseminations performed during no and mild heat stress (temperature-humidity index; THI< or =76) had greater conception rate (P<0.05; 38.8%) compared to AI performed during moderated heat stress conditions (THI>76; 17.6%). Number of mounts were higher for primiparous versus multiparous cows (P<0.05). Cows over 80 DIM during estrus exhibited fewer (P<0.05) standing events. The highest conception rate occurred with the combination of VO+HW, which confirms the premise that combination of multiple systems enhances both the efficiency and accuracy of estrus detection.

GFP labelling and epigenetic enzyme expression of bone marrow-derived mesenchymal stem cells from bovine foetuses.

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Since MSC from adult tissues represent a promising source of cells for a wide range of cellular therapies, there is high scientific interest in better understanding the potential for genetic modification and the mechanism underlying differentiation. The main objective of this study was to evaluate the potential for gene delivery using a GFP vector and lipofectamine, and to quantify the expression of epigenetic enzymes during foetal bMSC multilineage differentiation. Proportion of GFP-positive cells achieved (15.7% ± 3.5) indicated moderately low transfection efficiency. Analysis of DNA methyltransferase expression during MSC multilineage differentiation suggested no association with osteogenic and chondrogenic differentiation. However, up-regulation of KDM6B expression during osteogenic differentiation was associated with adoption of osteogenic lineage. Furthermore, increase in epigenetic enzyme expression suggested an intense epigenetic regulation during adipogenic differentiation.

Growth pattern of selected urban Chilean infants during exclusive breast-feeding.

This report describes the growth pattern of healthy, low middle and low socioeconomic class Chilean infants during exclusive breast-feeding. Two-hundred forty-two infants who were on exclusive breast-feeding at day 30 postpartum entered the study. Of these, 59% were fully nursing at 6 months and grew at a normal rate without receiving either supplementary milk or nondairy food. Supplementary feedings were administered in 27% of cases because of suspected primary inadequate milk output and in 14% of cases for other reasons such as pregnancy, illness, maternal work, or self-prescription. Full nursing provided the highest rate of weight increase during the first 3 months of life and a greater weight gain for boys than for girls up to the age of 6 months. Gastrointestinal pathology, malnourishment, or hospitalization were rare events in this population. It is concluded that maternal milk alone, if produced in sufficient amounts, can maintain normal growth up to the 6th month of life. The study supports the choice of exclusive breast-feeding on demand plus child growth monitoring up to 6 months over routine prescription of supplements at earlier times particularly where supplement administration fails to meet individual requirements.

PIP: This report describes the growth pattern of healthy, low middle and low socioeconomic class Chilean infants during exclusive breastfeeding. 242 infants who were on exclusive breastfeeding at day 30 postpartum entered the study. Of these, 59% were fully nursing at 6 months and grew at a normal rate without receiving either supplementary milk or nondairy food. Supplementary feedings were administered in 27% of the cases because of suspected primary inadequate milk output and in 14% of cases for other reasons such as pregnancy, illness, maternal work, or self prescription. Full nursing provided the highest rate of weight increase during the 1st 3 months of life and greater weight gain for boys than for girls up to the age of 6 months. Gastrointestinal pathology, malnourishment, or hospitalization were rare events in this population. It is concluded that maternal milk alone, if produced in sufficient amounts, can maintain normal growth up to the 6th month of life. The study supports the choice of exclusive breast feeding on demand plus child growth monitoring up to 6 months over routine prescription of supplements at earlier times particularly where supplement administration fails to meet individual requirements.

Subdermal contraceptive implants.

Subdermal contraceptive implants involve the delivery of a steroid progestin from polymer capsules or rods placed under the skin. The hormone diffuses out slowly at a stable rate, providing contraceptive effectiveness for 1-5 years. The period of protection depends upon the specific progestin and the type of polymer. Advantages of progestin implants include long term contraceptive action without requiring the user's or provider's attention, low dose of highly effective contraception without the use of estrogen, and fertility is readily reversible after the removal of implants. The levonorgestrel implant Norplant R system is the only one that has been approved for distribution. The contraceptive efficacy of Norplant is the highest observed amongst the most effective methods with an annual pregnancy rate of 0.2 during the first and second year and 1.1 on the fifth year. Menstrual problems are the main reason for the discontinuation of Norplant and 9% of women stopped using it during the first year of treatment. Other implants are still under development trying to simplify the method by reducing the number of units and to introduce other progestins that may minimize side effects. Norplant-2 was designed to release the same dose of progestin from only two covered rods. Evaluation of 1400 women enrolled, indicates that over 2 years the cumulative pregnancy rate is below 0.5 per 100 women. There are three single implants under development: Nestorone, 3-Keto-desogestrel and Uniplant that are expected to be effective for 1-2 years. Phase II clinical trials with Nestorone have been completed and no pregnancies have been observed in 1570 woman-months of use. Bleeding irregularities occurred in 20-30% of the women but there were only four terminations because of bleeding problems. A multricentric study is ongoing with a newly designed 3-keto-desogestrel implant named Implanon, which releases approx. 60 micrograms/day of the hormone. The objectives of this study are to assess contraceptive efficacy, safety and acceptability of Implanon. Another multricentric study is ongoing with Uniplant, which releases nomegestrol acetate with a duration of action for only 1 year. The objectives of the trial are to study the endocrine profile of Uniplant users and to evaluate the efficacy and acceptability of the method.

PIP: Subdermal contraceptive implants deliver progestin from polymer capsules or rods placed under the skin. Diffusing slowly from the polymer containers at a stable rate, the hormone provides contraception for 1-5 years, with the period of protection conferred dependent upon the specific progestin and type of polymer employed. Once inserted, the device allows a woman to have sexual intercourse over a certain period of time without any significant risk of becoming pregnant. Protection is ensured with a low drug dosage and no estrogen, and fertility is readily reversible once the implants are removed. The levonorgestrel implant Norplant R is the only subdermal contraceptive implant system approved for distribution. Annual pregnancy rates using Norplant are extremely low. Menstrual problems are the main reason why women discontinue using Norplant. Research is ongoing to reduce the number of implanted units and to introduce other progestins which may minimize side effects. Norplant-2 was designed to release the same dose of progestin from only two covered rods. Nestorone, 3-Keto-desogestrel, and Uniplant are single implants under development which are expected to be effective for 1-2 years. Completed phase II clinical trials with Nestorone found no pregnancies in 1570 woman-months of use, although bleeding irregularities occurred in 20-30% of women. A multicenter study is ongoing with a newly-designed 3-keto-desogestrel implant named Implanon, as well as another multicenter study with Uniplant, an implant which releases nomegestrol acetate with a one-year duration of action.

Lactational amenorrhea and the recovery of ovulation and fertility in fully nursing Chilean women.

The probability of experiencing the first postpartum bleeding, the first ovulation and the risk of pregnancy during exclusive breastfeeding was assessed in a selected group of urban Chilean women. Admission criteria included having had a normal pregnancy and a vaginal term delivery of a healthy infant and the desire to maintain breastfeeding for as long as possible. The risk of bleeding and the recovery of ovulation was assessed in 48 women selected for being amenorrheic and fully nursing at day 75 postpartum and their willingness to participate in the blood sampling protocol. The first bleeding and ovulation was experienced while fully nursing by 28% and 26% of these subjects, respectively, at day 180 postpartum. The probability of experiencing the first bleeding and the probability of pregnancy during full nursing were calculated for 236 women not contracepting who were enrolled during the first month postpartum. The cumulative probability of bleeding and of pregnancy was 52% and 9.4% at day 180 postpartum, respectively. The risk of pregnancy was less than 2% in the subset of amenorrheic cases. In this urban population selected for having the highest motivation and best breastfeeding performance, the association of breastfeeding with infertility was too weak to serve as an effective birth spacer, except for the period of lactational amenorrhea. When the first postpartum bleeding took place before the sixth postpartum month in fully nursing women, it had a good predictive value to indicate the onset of a higher risk period.

Fertility regulation in nursing women. IX. Contraceptive performance, duration of lactation, infant growth, and bleeding patterns during use of progesterone vaginal rings, progestin-only pills, Norplant implants, and Copper T 380-A intrauterine devices.

This study evaluated the performance of progesterone vaginal rings (n = 187), progestin-only pills (n = 117), Norplant implants (n = 120), and Copper T 380-A intrauterine devices (n = 122) in lactating women. Contraceptive efficacy, bleeding pattern, and influence of the method upon breastfeeding duration and infant growth were compared with those of untreated women (n = 236) who relied on lactational infertility. Participants were healthy, 18 to 38 years, had had a normal delivery, and were intending to breastfeed for as long as possible. Contraceptives were initiated at day 57 +/- 3 postpartum. Results are reported for the first year of use. All methods were highly effective, with pregnancy rates below 1%. None affected breastfeeding performance or the rate of infant growth. Users of the progestin-only methods experienced a period of lactational amenorrhea 4 to 5 months longer than did users of Copper T or untreated women. More than half of the women in each contraceptive group reported a bleeding in the first month after treatment initiation, which was not considered in the calculation of the duration of amenorrhea. Prolonged or frequent bleedings were infrequent. The proportion of bleedings lasting more than 10 days ranged from 0 in the progestin-only pills group to 7% in the Norplant implants group. The four methods, initiated around the eighth postpartum week, provided effective contraception with no negative effects upon lactation or infant growth and without the bleeding problems associated with their use in nonlactating women.

Fertility regulation in nursing women. VI. Contraceptive effectiveness of a subdermal progesterone implant.

Six progesterone pellets implanted subdermally were tested as contraceptive method for lactating women. One pregnancy was diagnosed in 1614 woman-months of observation, a failure rate which was similar to that observed in a contemporary group of Copper T users. Nineteen pregnancies were diagnosed in the 677 woman-months observed in untreated lactating women. The progesterone implants were effective when administered either at 30, 60 or 240 days after delivery. The duration of the effective life was 5 months and fertility was quickly restored afterwards. There were no deleterious effects upon maternal or infant health or upon lactation and the rate of child growth. The main problem encountered was the occurrence of pellet expulsion at a variable rate which appeared related to the manufacturing procedure.

Fertility regulation in nursing women: VII. Influence of NORPLANT levonorgestrel implants upon lactation and infant growth.

The objective of this study was to test the influence of NORPLANT implants upon lactation and infant growth. The results obtained during the first postpartum year are reported. One-hundred women were enrolled in the implant group. The control group was formed by 100 women who received a Copper T IUD. Treatments were administered at day 55 +/- 3. At admission all subjects were in exclusive breastfeeding with an adequate weight increase of the infant. No significant differences were found between the treated and control groups in the percentage of women in full nursing at different postpartum intervals, except for the 12th month postpartum where a lower percentage of cases from the NORPLANT group was in the full nursing category. No difference was found in the time of weaning. The infants showed a normal weight gain rate in both groups although the girls from the NORPLANT group showed a significantly lower daily weight increase during the 4th month than the girls from the IUD group. No pregnancies were diagnosed. No women asked for implant removal during the first year. No serious side effects related to treatment were detected. Bleeding irregularities were rare during breastfeeding which is in contrast with what occurs in non-nursing women treated with NORPLANT implants. Levonorgestrel concentrations found in milk were variable and individual values ranged from 23 to 311 pg/ml. The dose received by the infant can be estimated around 15 to 18 ng/Kg/day during the first month of treatment when the higher plasma values of levonorgestrel are observed. These levels is conjunction with the clinical observations reported here minimize the potential problems associated with levonorgestrel transference through maternal milk. Nevertheless, long-term studies are required to settle this matter and until more information is available, NORPLANT implant use in lactating women should be limited to cases who require a highly effective contraceptive method and where non-hormonal methods of similar effectiveness are contraindicated or unacceptable.

Fertility regulation in nursing women: VIII. Progesterone plasma levels and contraceptive efficacy of a progesterone-releasing vaginal ring.

The objectives of this trial were to test the contraceptive efficacy and measure progesterone plasma levels of women using progesterone-releasing rings during lactation. Two types of rings delivering an average of about 5 mg or 10 mg of progesterone per day were tested in 128 healthy nursing women. A control group was formed by 127 Copper T users. Rings were inserted at day 60 postpartum and replaced every 3 months with a new one. Initially progesterone plasma levels were around 10 nmol/l and 15 nmol/l for rings releasing 5 and 10 mg per day, respectively, and decreased slightly after 30 days. Levels observed in subsequent segments of use approximated those of the first segment. These levels are within the range shown to inhibit fertility in lactating women. One pregnancy was diagnosed in 739 woman-months of progesterone ring use, and none occurred in 794 woman-months of Copper T use, which contrasts with the high incidence of pregnancy in a group of untreated nursing women where 19 pregnancies resulted during 677 woman-months. No deleterious effects were detected lactation and infant growth or maternal and infant health. It is concluded that the vaginal rings releasing progesterone are a suitable contraceptive method for lactating women.

Fertility regulation in nursing women: III. Short-term influence of a low-dose combined oral contraceptive upon lactation and infant growth.

The study was designed to test the short-term influence of a low-dose combined oral contraceptive upon lactation and infant growth when treatment was initiated at day 30 postpartum. The contraceptive tested contained ethinyl estradiol 0.03 mg and levonorgestrel 0.15 mg. Two control groups were formed by women who received either an oral placebo or an injectable placebo at day 30 postpartum. An exacting list of requirements for admission and continuation in the study was applied to all groups. The oral contraceptive group had a lower percentage of cases in exclusive breastfeeding at day 91 postpartum when compared to both control groups. Infants in the oral contraceptive group showed a significantly lower average absolute weight at days 61 and 91 postpartum and a significantly lower average daily weight increase during the first month of treatment when compared to both control groups. It was concluded that the oral contraceptive tested had a negative influence upon lactation and child growth.

Fertility regulation in nursing women: IV. Long-term influence of a low-dose combined oral contraceptive initiated at day 30 postpartum upon lactation and infant growth.

The study was designed to test the long-term influence of a low-dose combined oral contraceptive upon lactation and infant growth when treatment was initiated at day 30 postpartum. The contraceptive tested contained ethinyl estradiol 0.03 mg and levonorgestrel 0.15 mg. Two control groups were formed by women who received an injectable placebo or a Copper T at day 30 postpartum. Women in the injectable placebo group received non-hormonal contraceptives at day 90 postpartum. An exacting list of requirements for admission and continuation in the study was applied to all groups. The oral contraceptive group had a significantly lower percentage of cases in full nursing from the 4th through the 10th postpartum month when compared to both control groups. The average absolute weight of infants in the oral contraceptive group was significantly lower at several ages when compared to the placebo group but not when compared to the Copper T group. No adverse side effects upon infant's health were detected. It was concluded that the oral contraceptive tested showed a moderate inhibitory influence upon lactation when treatment was initiated at the beginning of the second postpartum month.

Fertility regulation in nursing women: V. Long-term influence of a low-dose combined oral contraceptive initiated at day 90 postpartum upon lactation and infant growth.

The study was designed to test the long-term influence of a low-dose oral contraceptive upon lactation and infant growth when treatment was initiated at day 90 postpartum. The contraceptive tested contained ethinyl estradiol 0.03 mg and levonorgestrel 0.15 mg. The control group was formed by women who chose non-hormonal contraception at the same postpartum time. Only cases who were in exclusive breast-feeding with a minimal infant daily weight increase of 20 g per day during the third month of age were included. The oral contraceptive group showed a significantly lower percentage of cases in full nursing from the 6th postpartum month and a significantly lower infant weight increase during the 4th month of age when compared to the control group. No differences were found in the absolute infant weight up to one year of age. No adverse side effects upon infant's health were detected. It was concluded that the oral contraceptive tested showed a moderate inhibitory influence upon lactation when treatment was initiated at day 90 postpartum.

PIP: The influence of a low-dose oral contraceptive (OC) on lactation and child growth when treatment is initiated at day 90 postpartum in fully nursing women was tested on 59 women using OCs and with 82 fully breastfeeding women (controls) choosing nonhormonal contraception (Emko foam and Copper T IUDS). The 2 groups were similar in age, weight, hemoglobin, and parity. The OC group received 1 pill/day containing ethinyl estradiol .03 mg and levonorgestrel .15 mg for 21 days followed by a 1-week pill-free interval. Mothers were instructed to give no food, liquid or solid, up to the 6th postpartum month unless prescribed by the pediatrician. Follow-up visits were monthly through 6 months and bimonthly to the end of the year. Infant weight increase was used as the most important objective indicator of adequate milk output. The OC group showed a lower proportion of full nursing cases for all intervals after treatment and a higher proportion where supplement was administered either by medical prescription or maternal decision. No significant differences were found between the 2 groups in time of weaning or in average absolute weight of exclusively breastfed infants during the 1st year. During the 1st month of treatment, infants in the OC group experienced a significantly lower weight increase than children in the control group. Total increase to the 6th month of age was 4881 +or- 939 g in the OC group and 5005 +or- 657 in the control group. An inhibitory effect of the low dose OC was deduced from the significantly lower percentage of cases in full nursing after initiation of treatment and from the significantly lower infant weight increases during the 1st month of pill use. The authors concluded that OCs do not represent a good choice during breastfeeding.