Hypoxia limits differentiation and up-regulates expression and activity of prostaglandin H synthase 2 in cultured trophoblast from term human placenta

OBJECTIVE: We determined the effect of hypoxic conditions on cellular differentiation and prostaglandin H synthase expression in cultured human term trophoblast. STUDY DESIGN: Cytotrophoblasts isolated from term placentas were cultured for 24-72 hours in a standard (20% oxygen) or hypoxic (1% to 2% oxygen) atmosphere. Trophoblast biochemical differentiation was determined by release of human chorionic gonadotropin. Morphologic differentiation was evaluated by epifluorescent and confocal microscopic examination of cultures after dual cytochemical staining for surface membrane desmosomes and intracytoplasmic nuclei. Prostaglandin H synthase 2 expression was determined by Western blot analysis and correlated with enzyme activity by immunoassay of 2 prostaglandin H synthase 2 products, prostaglandin E2 and thromboxane. RESULTS: Human chorionic gonadotropin levels in media and syncytiotrophoblast formation were markedly diminished in trophoblast cultured in hypoxia, compared with trophoblast in control cultures, whereas viability was unchanged. Hypoxia up-regulated expression of trophoblast prostaglandin H synthase 2 but not of prostaglandin H synthase 1 and increased prostaglandin E2 and thromboxane release. CONCLUSIONS: Hypoxia limits differentiation and enhances prostaglandin H synthase 2 expression in cultured villous trophoblast. These responses may account for the cytotrophoblast prominence characteristic of placentas exposed to attenuated oxygen delivery.     

A novel change in cytologic localization of human chorionic gonadotropin and human placental lactogen in first-trimester placenta in the course of gestation.

Cytologic localization of human chorionic gonadotropin and human placental lactogen in developing human early placenta was analyzed by avidin-biotin immunoperoxidase techniques with an affinity-purified polyclonal antibody to beta-human chorionic gonadotropin carboxyl terminal peptide and a polyclonal antibody to human placental lactogen. In 4- to 5-week placentas human chorionic gonadotropin and human placental lactogen were found to be primarily localized to cytotrophoblasts, whereas in 6- to 12-week placentas these substances were exclusively localized to syncytiotrophoblast. We previously reported that a similar change in cytologic localization of epidermal growth factor and its receptor from cytotrophoblasts to syncytiotrophoblast in first-trimester placenta appeared between 5 and 6 weeks of gestation. Because epidermal growth factor was demonstrated to stimulate human chorionic gonadotropin and human placental lactogen production by early placental tissues, their simultaneous expression, as well as epidermal growth factor and its receptor in the cytotrophoblast of 4- to 5-week placenta and in the syncytiotrophoblast of 6- to 12-week placenta, implies that human chorionic gonadotropin and human placental lactogen production by first-trimester placenta may be regulated in an autocrine manner, wherein epidermal growth factor may serve as the signal. These findings suggest that in very early placenta, before 6 weeks of gestation, no sequential expression of human chorionic gonadotropin and human placental lactogen closely linked to syncytia formation may exist and that both can be expressed in the cytotrophoblast or undifferentiated stem cell of villous trophoblast in very early placenta.     

Human chorionic gonadotropin exhibits potent inhibition of preterm delivery in a small animal model.

OBJECTIVE: The purpose of this study was to test the capability of human chorionic gonadotropin to inhibit prostaglandin-induced preterm delivery in a murine model. STUDY DESIGN: A preterm delivery model was developed by using intraperitoneal injection of 20 microgram of prostaglandin F(2)(alpha) to induce preterm labor in C3H/HeN inbred mice. Mice were then pretreated with human chorionic gonadotropin 4 hours before administration of prostaglandin F(2)(alpha), and time to delivery of the first pup was recorded. After initial promising results, mice were then given increasing intraperitoneal doses of human chorionic gonadotropin (100 IU, 250 IU, or 1000 IU or sodium chloride solution vehicle) 4 hours after administration of prostaglandin F(2)(alpha). The specificity of the human chorionic gonadotropin effect was assessed by treating mice with whole human chorionic gonadotropin, an equal mass dose of the beta-subunit or the alpha-subunit of human chorionic gonadotropin, or an equal mass dose of luteinizing hormone 4 hours after administration of prostaglandin F(2)(alpha). Delivery times between groups were compared by using the Mann-Whitney U test and the log-rank test. Survival estimates were computed by using the Kaplan-Meier method. RESULTS: Pilot studies in 52 mice confirmed that a single intraperitoneal injection of 20 microgram of prostaglandin F(2)(alpha) on day 16 (80% gestation) consistently induced preterm delivery compared with the effect of sodium chloride solution on control mice (prostaglandin F(2)(alpha), 19.3 +/- 2.9 hours; sodium chloride solution, 53.5 +/- 13.6 hours; P <.0001). Mice pretreated with human chorionic gonadotropin (1000 IU) demonstrated significant delays in delivery times compared with the prostaglandin-only group (prostaglandin F(2)(alpha) only, 21.9 +/- 2. 0 hours; human chorionic gonadotropin pretreatment plus prostaglandin F(2)(alpha), 48.5 +/- 20 hours; P <.0001; n = 17). Mice treated with human chorionic gonadotropin (100 IU, 250 IU, 1000 IU) 4 hours after administration of prostaglandin F(2)(alpha) demonstrated significant dose-dependent inhibition of preterm delivery compared with the prostaglandin-only group (P <.00005; n = 34). Mice treated with the alpha-subunit or the beta-subunit of human chorionic gonadotropin after prostaglandin administration did not demonstrate delays in delivery times (P =.46; n = 27). Administration of luteinizing hormone delayed delivery compared with the effect of prostaglandin F(2)(alpha) on control animals (P <.05; n = 17); however, the effect was less pronounced than that seen with a mass equivalent of human chorionic gonadotropin. CONCLUSIONS: Human chorionic gonadotropin exhibits potent inhibition of prostaglandin-induced preterm delivery in mice. The effect is dose-dependent, and whole human chorionic gonadotropin is required to elicit inhibition. Further studies are needed to determine the safety and efficacy of human chorionic gonadotropin as a potential therapy for preterm labor inhibition in human pregnancy.     

Elevated maternal midtrimester serum free beta-human chorionic gonadotropin levels in vegetarian pregnancies that cause increased false-positive Down syndrome screening results

OBJECTIVE: The aim of this study was to examine whether midtrimester maternal serum free beta-human chorionic gonadotropin and alpha-fetoprotein levels for Down syndrome screening differed in vegetarian pregnancies and omnivore pregnancies and to evaluate whether maternal serum vitamin B(12) concentration affected these maker levels. STUDY DESIGN: Ninety-eight vegetarian and 122 omnivore singleton pregnancies were studied. Reference levels of free beta-human chorionic gonadotropin and alpha-fetoprotein were based on a population of 6312 singleton euploid pregnancies that had been surveyed previously. Serum free beta-human chorionic gonadotropin and alpha-fetoprotein levels were measured by enzyme immunoassay or radioimmunoassay. Multiples of the median values were calculated to determine whether different diet habits affected serum biomarker levels. Maternal serum vitamin B(12) levels were determined with radioimmunoassay. RESULTS: The free beta-human chorionic gonadotropin multiples of the median values were elevated significantly in the vegetarian pregnancies group (1.28 multiples of the median) compared with that of the reference population (1.00 multiples of the median) (P<.001). A negative association between the serum free beta-human chorionic gonadotropin multiples of the median values and the concentration of maternal serum vitamin B(12) was observed in the vegetarian pregnancies. No correlation was found between the alpha-fetoprotein multiples of the median values and the maternal serum vitamin B(12) concentration. CONCLUSION: The current data showed that the midtrimester maternal serum free beta-human chorionic gonadotropin levels increased in vegetarian pregnancies and led to an elevated false-positive rate in screening for Down syndrome compared with pregnant women with regular diet and resulted in unnecessary invasive procedures. It is necessary to establish vegetarian pregnancy alpha-fetoprotein and beta-human chorionic gonadotropin reference levels to correct increased false-positive screening results.     

Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.

OBJECTIVE: We evaluated the value of all 3 common biochemical serum markers, maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, and unconjugated estriol, and combinations thereof as predictors of pregnancy outcome. STUDY DESIGN: A total of 60,040 patients underwent maternal serum screening. All patients had maternal serum alpha-fetoprotein measurements; beta-human chorionic gonadotropin was measured in 45,565 patients, and 24,504 patients had determination of all 3 markers, including unconjugated estriol. The incidences of various pregnancy outcomes were evaluated according to the serum marker levels by using clinically applied cutoff points. RESULTS: In confirmation of previous observations, increased maternal serum alpha-fetoprotein levels (>2.5 multiples of the median) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, intrauterine growth restriction, intrauterine fetal death, oligohydramnios, and abruptio placentae. Increased beta-human chorionic gonadotropin levels (>2.5 multiples of the median [MoM]) were significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, and intrauterine fetal death. Finally, decreased unconjugated estriol levels (<0.5 MoM) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, intrauterine growth restriction, and intrauterine fetal death. As with increased second-trimester maternal serum alpha-fetoprotein levels, increased serum beta-human chorionic gonadotropin and low unconjugated estriol levels are significantly associated with adverse pregnancy outcomes. These are most likely attributed to placental dysfunction. CONCLUSION: Multiple-marker screening can be used not only for the detection of fetal anomalies and aneu-ploidy but also for detection of high-risk pregnancies.     

Accuracy of serum beta-human chorionic gonadotropin cutoff values at 42 and 49 days' gestation

OBJECTIVE: The accuracy of serum beta-human chorionic gonadotropin levels as cutoff values for estimating gestational age was studied. MATERIAL AND METHODS: A database was created using information from previously performed research studies, which allowed entry of women both less than and greater than 49 days' gestation, involving medical abortion. Serum beta-human chorionic gonadotropin determinations and vaginal ultrasonography were performed in all studies before treatment. A total of 574 women had data available for analysis. A receiver operating characteristic curve was created to evaluate the predictive value of potential beta-human chorionic gonadotropin cutoff values for 42 and 49 days' gestation. RESULTS: Appropriate serum beta-human chorionic gonadotropin cutoff values for 42 and 49 days' gestation were 23,745 mIU/mL (sensitivity, 96%; specificity, 91%; positive predictive value, 68%; negative predictive value, 99%) and 71,160 mIU/mL (sensitivity, 95%; specificity, 62%; positive predictive value, 76%; negative predictive value, 91%), respectively. Under 42 days' gestation, the serum beta-human chorionic gonadotropin-time relationship appears to be linear, with a greater diversity of individual values after 42 days. CONCLUSION: Serum beta-human chorionic gonadotropin values can be used with reasonable accuracy to screen for a gestational age up to 49 days' gestation.     

Disappearance of human chorionic gonadotropin after cesarean section with regard to fetal sex

BACKGROUND: To evaluate the influence of gender on the disappearance of human chorionic gonadotropin by cesarean section after fullterm pregnancies. MATERIALS AND METHODS: Forty-nine uncomplicated pregnancies: 26 had male (male group) and 23 had female (female group) fetuses. RESULTS: Before the cesarean section the serum human chorionic gonadotropin levels were higher in the female than in the male bearing pregnancies. After cesarean section the human chorionic gonadotropin levels fell rapidly. The decrease in the human chorionic gonadotropin values was significantly faster in the male than in the female group during the first hours after delivery (2P < 0.02), while no significant difference was seen after 24 and 72 h. CONCLUSION: This study shows a significantly faster human chorionic gonadotropin disappearance rate in pregnancies with male compared with female fetuses during the first hours after a cesarean section. This indicates a gender difference, which could be related to different human chorionic gonadotropin molecular structures or to more specific metabolic events.     

A high-grade uterine leiomyosarcoma with human chorionic gonadotropin production.

Ectopic human chorionic gonadotropin production has been described in a wide variety of non-germ cell tumors, particularly in epithelial tumors, but rarely in sarcomas. In this report, we describe the case of 49-year-old woman with a history of "uterine fibroids," who presented with vaginal bleeding and a positive urine pregnancy test. After pregnancy was ruled out by ultrasound, the patient underwent a laparotomy and hysterectomy for a presumptive diagnosis of "fibroids" and was found to have carcinomatosis at the time of the surgery. Therefore optimal debulking of tumors was performed. Two weeks later, the patient developed a small bowel obstruction, which apparently was due to rapid recurrence of tumors in the abdomen, and soon afterwards she died. Microscopically, the resected pelvic mass was composed of highly atypical and pleomorphic spindle cells admixed with many multinucleated giant cells. The tumor had a high mitotic rate along with areas of hemorrhage and necrosis. Immunohistochemically, the tumor cells were positive for vimentin, desmin, smooth muscle actin, and beta-human chorionic gonadotropin, and were negative for epithelial membrane antigen, keratin AE1/3, S-100, CD31, CD117, Ber-EP4, WT-1, estrogen and progesterone receptors. The majority of cells, including the multinucleated giant cells, were strongly immunoreactive for beta-human chorionic gonadotropin. Only three cases of leiomyosarcomas with beta/human chorionic gonadotropin production have been described in the literature, and all three cases had extrauterine origin. Our case, to the authors' best knowledge, is the first uterine leiomyosarcoma with prominent beta/human chorionic gonadotropin production.     

Epithelioid trophoblastic tumor of the endocervix: a case report

BACKGROUND: It is difficult to recognize epithelioid trophoblastic tumor (ETT) as a trophoblastic disease because of its rarity and growth pattern simulating a carcinoma. CASE REPORT: A 36-year-old woman with stage IB(1) squamous cell carcinoma of the uterine cervix and a high serum beta-human chorionic gonadotropin (beta-hCG) level underwent radical hysterectomy with pelvic and para-aortic lymphadenectomy. However, light microscopic findings and immunohistochemical studies with pan-cytokeratin, epithelial membrane antigen, inhibin-alpha, beta-hCG, and human placental lactogen revealed ETT of the endocervix. The patient is alive with no evidence of disease 12 months after surgery. CONCLUSION: Before the patient is resorted to radical surgical interventions for assumed cervical carcinoma, ETT should be ruled out in women of reproductive age with endocervical tumors and elevated serum beta-hCG levels.     

Effects of melatonin on progesterone production by human granulosa lutein cells in culture.

OBJECTIVE: To test the hypothesis that melatonin modulates steroid synthesis in the human ovary. DESIGN: Granulosa lutein cells obtained from in vitro fertilization cycles were cultured in medium containing melatonin and human chorionic gonadotropin (hCG). RESULTS: Progesterone (P) secretion by granulosa lutein cells increased progressively in both basal and hCG-stimulated conditions, up to 96 hours in culture, plateaued at 144 and decreased thereafter. Melatonin (10(-7), 10(-9), 10(-11) M) had no effect on basal P or 17 beta-estradiol production. The addition of melatonin to the hCG-treated granulosa lutein cells significantly (P less than 0.05) potentiated the stimulatory effect of hCG on P production. The effect was most prominent after 144 and 196 hours of incubation. CONCLUSION: This observation suggests a role for melatonin in the intraovarian control of P production in the human ovary.     

Expression of mitochondria-dependent apoptosis genes (p53, Bax, and Bcl-2) in rat granulosa cells during follicular development

OBJECTIVE: We examined rat ovarian granulosa cells at different follicular stages and evaluated the apoptosis pattern of the mitochondria-dependent genes during folliculogenesis. METHODS: After down-regulating ovarian function with gonadotropin-releasing hormone agonist (GnRHa), granulosa cells were collected from the rat ovary at different stages of the following different hormonal treatment paradigms: stage E (after estrogen treatment), EF (after E + follicle-stimulating hormone [FSH] treatment), and EF hCG (after E + FSH + human chorionic gonadotropin treatment). To evaluate the in vitro susceptibility of granulosa cells at different developmental stages to apoptosis, the collected cells were cultured in a serum-free medium with or without E2 for 24 hours. The regulation of apoptosis in the granulosa cells was analyzed using fluorescein-activated cell sorting, quantitative competitive polymerase chain reaction, and western blot methods. RESULTS: The apoptosis rate of the freshly isolated granulosa cells tended to increase according to the hormonal treatment paradigm. In addition, during the hormone treatment, mitochondria-dependent apoptosis genes showed the following changes: although the Bax mRNA level did not change, the Bcl-2 mRNA level decreased significantly (P <.05). The p53 mRNA level increased significantly (P <.05) and closely matched the apoptosis rate (R = 0.7, P <.05). The expression of the active form of the caspase-3 protein (the final executioner of cell death) tended to increase and showed a good correlation with the apoptosis rate (R = 0.96, P <.01). After an in vitro culture of the granulosa cells, the apoptosis rate tended to increase at all stages, particularly stage EF hCG (P <.05). Bax and p53 mRNA tended to increase and showed a good correlation with the apoptosis rate (R = 0.64, P <.05 and 0.86, P <.01). The Bcl-2 mRNA level tended to decrease at all stages showing no correlation with the apoptosis rate. The expression level of the active caspase-3 protein tended to increase at all stages and showed a good correlation with the apoptosis rate (R = 0.93, P <.01). CONCLUSION: Apoptosis of rat ovarian granulosa cells tends to increase according to the stage of follicular development. Among the mitochondria-dependent genes, p53 closely correlates with granulosa cell apoptosis during follicular development.     

Characterization of pure human first-trimester cytotrophoblast cells in long-term culture: growth pattern, markers, and hormone production

Pure long-term cytotrophoblast cultures were established from human first-trimester placentas by growing chorionic villus explants without enzymatic digestion. Cytoplasmic human chorionic gonadotropin was detectable in all (100%) cells in culture when labeled with a polyclonal anti-human chorionic gonadotropin antibody and in 71% to 83% of cells labeled with a monoclonal anti-alpha-human chorionic gonadotropin antibody. Most of the cells expressed cytokeratin and surface Trop-1 and Trop-2 antigens (89% to 95%), but none expressed cytoplasmic vimentin or surface 63D3 antigens. Study of the ultrastructure of the cells demonstrated epithelial morphologic features. The average doubling time of the trophoblast was 48 to 96 hours. Some of the lines have been continuously propagated for 8 months. They produced variable amounts of human chorionic gonadotropin (50 to 710 mIU/ml per 10(5) cells per 24 hours). The basal level of progesterone secreted by trophoblast (444.4 +/- 32.4 pg/ml per 10(5) cells per 24 hours) doubled in the presence of pure human chorionic gonadotropin (100 ng/ml). They produced small amounts of 17 beta-estradiol (less than 20 pg/ml per 10(5) cells per 24 hours); human chorionic gonadotropin had no effect on the estradiol production. Trophoblast-derived human chorionic gonadotropin acted as a growth factor because trophoblast proliferation (measured by uptake of thymidine labeled with tritium) was reduced by 60% in the presence of an anti-human chorionic gonadotropin antibody. Availability of pure, functionally competent human cytotrophoblast in long-term cultures is relevant for further studies in reproduction biology.     

Modulation of trophoblast function by tumor necrosis factor-alpha: a role in pregnancy establishment and maintenance?

OBJECTIVE: Trophoblast differentiation is a critical process for successful implantation and establishment of the human placenta. The aim of this study was to characterize the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) on the expression of markers of trophoblast function and differentiation.STUDY DESIGN: Human cytotrophoblasts were stimulated with 1 and 10 ng/mL recombinant TNF-alpha or IL-6. Cell viability was determined and conditioned culture media was analyzed by gelatin zymography to assess protease secretion and by enzyme-linked immunosorbent assays to measure production of beta-human chorionic gonadotropin and oncofetal fibronectin. RESULTS: TNF-alpha increased secretion of urokinase-type plasminogen activator up to 3-fold of basal unstimulated production. Stimulation of cytotrophoblasts with this cytokine also inhibited beta-human chorionic gonadotropin secretion up to 75%. TNF-alpha did not modify the secretion of matrix metalloproteinase-9 and oncofetal fibronectin. IL-6 had no effect on these trophoblast differentiation markers. CONCLUSION: These results show that TNF-alpha stimulated cytotrophoblasts modulate the expression of differentiation markers, down-regulating the autocrine signals that promote syncytialization, and increasing their invasive capacity through up-regulation of proteases. We suggest that this regulatory mechanism of trophoblast function could play an important role during trophoblast implantation, in pregnancy failure and in the normal and pathologic rupture of fetal membranes.     

Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies

OBJECTIVE: The purpose of this study was to examine the clinical significance of high maternal serum human chorionic gonadotropin levels in the second trimester in singleton and twin pregnancies within the Ontario maternal serum screening program. STUDY DESIGN: The study group comprised 564 women with singleton pregnancies with total maternal serum human chorionic gonadotropin levels of > or =4.0 multiples of the median (MoM) and serum marker alpha-fetoprotein levels of <2.0 MoM. The cases were matched with 1692 control subjects who had both serum marker alpha-fetoprotein levels and maternal serum human chorionic gonadotropin levels of <2.0 MoM. The second part of the study comprised 93 twin pregnancies with maternal serum human chorionic gonadotropin levels of > or =5.0 MoM and serum marker alpha-fetoprotein levels of <4.0 MoM; the control group (n = 1496) had serum marker alpha-fetoprotein levels of <4.0 MoM and maternal serum human chorionic gonadotropin levels of <5.0 MoM. The final part of the study included 25 women with extremely high maternal serum human chorionic gonadotropin levels (> or = 14;10 MoM). RESULTS: Of the singleton pregnancies with maternal serum human chorionic gonadotropin levels of > or = 14;4.0 MoM, 22.5% had severe adverse obstetric outcomes, compared with only 10.9% of the matched control population (P =.001). Women with markedly elevated maternal serum human chorionic gonadotropin levels had significantly increased risks of having spontaneous miscarriage, small-for-gestational-age infants, pregnancy-associated hypertensive disorder, and preterm delivery. Of the women with twin pregnancies with high maternal serum human chorionic gonadotropin levels (> or =5.0 MoM), 71% had at least one complication (such as miscarriage and preterm delivery) compared with 55.3% in the control group. Finally, 23 of 25 women with extremely high maternal serum human chorionic gonadotropin levels (> or = 14;10 MoM) had serious adverse outcomes (such as fetal abnormalities, pregnancy-associated hypertensive disorder, premature separation of placenta, intrauterine growth restriction, neonatal respiratory distress syndrome, and neonatal jaundice). CONCLUSION: Pregnancies with an elevated maternal serum human chorionic gonadotropin level are associated with adverse obstetric outcomes. Increased maternal and fetal surveillance is warranted in these pregnancies.     

Interleukin-1: local production and modulation of human granulosa luteal cells steroidogenesis.

OBJECTIVES: To investigate the possibility of local interleukin-1 (IL-1) and IL-1 inhibitor production by human granulosa and cumulus cells and to assess their direct effects on the steroidogenesis of these cells in vitro. DESIGN: Prospective study. PARTICIPANTS: Normal ovulatory women undergoing ovulation induction for in vitro fertilization. INTERVENTION: Pretreatment of patients with gonadotropin-releasing hormone analogue, human menopausal gonadotropin, and human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURES: Retrieval and isolation of granulosa luteal cells and follicular fluid (FF). Granulosa luteal cells and cumulus cells cultured and analyzed by fluorescent activated cell sorter. Follicular fluid separated and bioassayed for IL-1 and IL-1 inhibitory activity. Steroid measurement performed. Interleukin-1 inhibitor purified. Interleukin-1 and IL-1 inhibitor bioassay performed. Statistical analysis made and interpreted. RESULTS: Interleukin-1, but not IL-1 specific inhibitory activity, was found in granulosa and cumulus cell cultures and also in FF, only after its purification on a high-pressure liquid chromatography column. Under nonstimulated conditions, neither IL-1 nor IL-1 inhibitor had any effect on basal progesterone (P) or estradiol (E2) secretion. However, IL-1 inhibitor demonstrated significant (P < 0.01) inhibition of hCG-stimulated P secretion (from 200 to 110 ng/10,000 cells per 24 hours). In addition, IL-1 demonstrated a significant (P < 0.05) and dose-dependent inhibition of hCG-stimulated E2 production (from 6,832 +/- 460 to 4,237 +/- 141 pg/10,000 cells per 24 hours). CONCLUSIONS: Interleukin-1 may exert a significant local autocrine regulatory role in the human ovary.     

Effects of fetal sex, stage of gestation, dibutyryl cyclic adenosine monophosphate, and gonadotropin releasing hormone on secretion of human chorionic gonadotropin by placental explants in vitro

Explants from 16 term and 6 midtrimester placentas were cultured for 6 days. Statistically significant increases in secretion of human chorionic gonadotropin occurred in control medium cultures of both term and midtrimester explants during the 6-day culture period (p less than 0.01). Statistically significant increases in secretion of human chorionic gonadotropin were produced by 2 mmol/L dibutyryl cyclic adenosine monophosphate in both the term (p less than 0.01) and the midtrimester (p less than 0.01) explants. There was no effect of gonadotropin releasing hormone. The ratio of human chorionic gonadotropin secretion from midtrimester explants to that from term explants varied under different conditions, dropping from twentyfold in day 1 cultures to elevenfold for maximum secretion produced after culture in control medium for up to 6 days. A further drop in the ratio to fourfold was observed for the maximal response to 2 mmol/L dibutyryl cyclic adenosine monophosphate treatment. Explants from term female infants produced significantly more human chorionic gonadotropin than those from term male infants (p less than 0.05), but the sex difference disappeared after stimulation with 2 mmol/L dibutyryl cyclic adenosine monophosphate.     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.     

Synthesis of placental proteins by the human placenta perfused in vitro. Preliminary report

Fresh human placental tissue from deliveries at term was perfused in closed circulation on both the maternal and fetal side. At given times during the 4-hour perfusion period, the amount of human chorionic gonadotropin, human placental lactogen, pregnancy-specific beta 1-glycoprotein and pregnancy-associated plasma protein A was determined in the perfusing buffer. These placental proteins were also quantified in the placental tissue before and after perfusion. Calculations showed that all placentae (n = 3) were able to synthesize all 4 proteins tested.     

Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation

Cytologic localization of epidermal growth factor receptor and myc oncogene protein product in developing human placenta was analyzed by avidin/biotin immunoperoxidase techniques with a monoclonal antibody to epidermal growth factor receptor and an affinity purified polyclonal antibody to myc protein product. Epidermal growth factor receptor was found to be almost exclusively localized to syncytiotrophoblast, paralleling the immunohistochemical localization of human chorionic gonadotropin. Since epidermal growth factor has been shown to stimulate human chorionic gonadotropin and human placental lactogen production by cultured early placental tissue, the present finding that epidermal growth factor receptor was localized to mitotically inactive syncytiotrophoblast suggests a role for epidermal growth factor receptor in the induction of differentiated function of trophoblast rather than trophoblast proliferation. By contrast, myc protein product was found to be predominantly localized to cytotrophoblastic cells, paralleling the autoradiographic distribution of replicating cytotrophoblast identified by tritiated thymidine labeling of placental explant. A close similarity between the cytologic localization of myc protein product and tritiated thymidine labeling of placental explant suggests that myc protein expression is linked to trophoblast proliferation. Furthermore, immunohistochemical cellular levels of both epidermal growth factor receptor and myc protein product were most pronounced in early placenta and declined in term placenta. Thus myc protein product and epidermal growth factor receptor seem to play a crucial role in the induction of trophoblast proliferation and differentiation, respectively, during the development of human placenta.     

Trophic effects of first-trimester human trophoblasts and human chorionic gonadotropin on lymphocyte proliferation

We examined the effects of pure first-trimester human trophoblast cells grown in long-term cultures or their secreted products on the proliferation of human peripheral blood lymphocytes cultured alone, under allogeneic stimulation, or in the presence of concanavalin A. Both trophoblasts and their culture supernatants stimulated lymphocyte proliferation. Culture supernatant had a moderate enhancing effect on lymphocyte mitogenesis in mixed lymphocyte cultures and in the presence of concanavalin A. Anti-human chorionic gonadotropin antibody suppressed the proliferative effect of trophoblast cells and their supernatants in the above experiments in a dose-dependent manner. At physiologic concentrations, both pure and impure forms of human chorionic gonadotropin enhanced (in a dose-dependent manner) proliferation of lymphocytes cultured alone (peak stimulation index, 6 to 7.8 at approximately 7 IU/ml). At higher concentrations (20 to 400 IU/ml) the proliferative effect was abolished. Trophoblast culture supernatant induced the expression of interleukin-2 receptors on lymphocytes after 48 hours of incubation. The supernatant also stimulated proliferation of human colon carcinoma cells. Thus trophoblast or trophoblast-derived human chorionic gonadotropin has a lymphocytotrophic function that may have implications for fetal survival.