Gonadotrophin stimulation in IVF alters the immune cell profile in follicular fluid and the cytokine concentrations in follicular fluid and serum.

Study question: Are the immune cell profiles and the cytokine concentrations in follicular fluid (FF) and serum at the preovulatory stage different in conventional exogenous gonadotrophin stimulated IVF (c-IVF) compared with natural cycle IVF (NC-IVF)? Summary answer: The cell counts of CD45+ leucocytes and T cell subpopulations and the cytokine concentrations in FF and serum are different in c-IVF compared to NC-IVF. What is known already: FF-derived cells are heterogeneous. Immune cells are involved in intra-ovarian processes and cytokines are required for normal follicular development. Gonadotrophins stimulate the regulatory intrafollicular system and influence the local distribution of immune cells and the intrafollicular release of cytokines. Administration of exogenous gonadotrophins may have a significant effect on this local regulatory system, which then in turn could influence oocyte quality. Study design, size, duration: The study included 105 patients, 69 undergoing c-IVF and 36 undergoing NC-IVF. c-IVF was performed by exogenous ovarian stimulation with hMG and GnRH antagonists. Participants/materials, setting, methods: FF samples were collected from the first dominant follicle in c-IVF without pooling and from single leading preovulatory follicles in NC-IVF. Three different approaches were used to analyze FF samples: (i) microscopic investigation of CD45+ leucocytes, (ii) fluorescence-activated cell sorting to determine CD19+ B cells and CD3+ T cells including T cell subpopulations (CD4+, CD8+), and (iii) evaluation of tumour necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), interleukins (IL)-2, -6, -8, -10 and vascular endothelial growth factor (VEGF) levels in matched FF and serum samples using the Bio-Plex® platform. Main results and the role of chance: FF obtained from c-IVF contained proportionally more CD45+ leucocytes (P = 0.0384), but fewer CD8+ cytotoxic T cells than FF from NC-IVF. CD3+ T lymphocytes were the most common type of lymphocytes, and the number thereof was comparable in the two study groups. In c-IVF, serum VEGF levels were higher (P = 0.007) than in NC-IVF while FF contained marginally decreased concentrations of IL-8 in c-IVF in comparison to NC-IVF. The cytokine concentration gradient between FF and serum in c-IVF was 10-fold for IL-8 and 8-fold for VEGF and thereby markedly lower than in NC-IVF, where the differences were 32-fold and 30-fold, respectively. Strong positive correlations were determined between FF- IL-10 and FF- VEGF in c-IVF (r = 0.85, P < 0.0001) and in NC-IVF (r = 0.81, P < 0.0001). Large scale data: N/A. Limitations, reasons for caution: The ovulation of NC-IVF follicles was induced by the exogenous administration of hCG, which means that the environment did not fully correspond to the physiological situation. Wider implications of the findings: The differences in the immune profile and the cytokine concentrations in c-IVF and NC-IVF follicles support the hypothesis that conventional ovarian stimulation affects indirectly and heterogeneously the intrafollicular milieu, and thereby possibly affects the oocyte quality and the IVF outcome. However, further studies are needed to confirm our findings and to refine stimulation protocols in the context of optimizing the intrafollicular environment during oocyte maturation. Study funding/competing interest(s): The study was supported by a research grant from IBSA Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. The authors are clinically involved in low dose mono-follicular stimulation and IVF-therapies, using gonadotrophins from all gonadotrophins distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH.

Intrafollicular inflammatory cytokines but not steroid hormone concentrations are increased in naturally matured follicles of women with proven endometriosis.

PURPOSE: The aim of this study was to assess whether the intrafollicular cytokine profile in naturally developed follicles is different in women with endometriosis, possibly explaining the lower reproductive outcome in endometriosis patients. METHODS: A matched case-control study was conducted at a university-based infertility and endometriosis centre. The study population included 17 patients with laparoscopically and histologically confirmed endometriosis (rAFS stages II-IV), each undergoing one natural cycle IVF (NC-IVF) treatment cycle between 2013 and 2015, and 17 age-matched NC-IVF women without diagnosed endometriosis (control group). Follicular fluid and serum was collected at the time of follicle aspiration. The concentrations of inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-15, IL-18, TNF-α) and hormones (testosterone, estradiol, AMH) were determined in follicular fluid and serum by single or multiplexed immunoassay and compared between both groups. RESULTS: In the follicular fluid, IL-1ß and IL-6 showed significantly (P < 0.001 and 0.01, respectively) higher median concentrations in the endometriosis group than in the control group and a tendency towards endometriosis severity (rAFS stage) dependence. The levels of the interleukins detectable in follicular fluid were significantly higher than those in the serum (P < 0.01). Follicular estradiol concentration was lower in severe endometriosis patients than in the control group (P = 0.036). Follicular fluid IL-1ß and IL-6 levels were not correlated with estradiol in the same compartment in neither patient group. CONCLUSIONS: In women with moderate and severe endometrioses, some intrafollicular inflammatory cytokines are upregulated and not correlated with intrafollicular hormone concentrations. This might be due to the inflammatory microenvironment in endometriosis women, affecting follicular function and thereby possibly contributing to the reproductive dysfunction in endometriosis.

Dienogest mediates midkine suppression in endometriosis.

STUDY QUESTION: What are the effects of dienogest (DNG) on midkine (MK) production in women with endometriosis? SUMMARY ANSWER: DNG-mediated down-regulation of MK in vivo and in vitro. WHAT IS KNOWN ALREADY: DNG is an oral progestin that alleviates painful symptoms of women with endometriosis with a favourable tolerability and safety profile. Its effects on MK, a growth factor that plays an important role in endometriosis, have not yet been investigated. STUDY DESIGN, SIZE, DURATION: Prospective in vivo study on 283 patients subjected to laparoscopy for benign pathologies in a University hospital and in vitro cultures of primary endometrial stromal cells (ESC) from 6 of these women with histologically confirmed endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: MK concentrations in the peritoneal fluid (PF) of women were measured by ELISA and compared based on endometriosis status and the use of DNG. A subsequent in vitro analysis with ESC was used to confirm the direct influence of DNG and other progestins including, norethisterone acetate (NETA) and medroxyprogesterone acetate (MPA) on MK mRNA production. MAIN RESULTS AND THE ROLE OF CHANCE: The final study population consisted of 253 women. Of these, 165 suffered from endometriosis, with 62 of them taking DNG (DNG group) and 103 taking no hormone treatment (non-DNG group) during at least 3 months before surgery. Another 88 women were endometriosis free (non-endometriosis group). The concentration of MK was highest in the PF of women in the non-DNG group (median 5.26 ng/ml, IQR 2.74-8.46). Significantly lower concentrations were found in the non-endometriosis group (median 3.51 ng/ml, IQR: 1.90-7.53, P = 0.028). The lowest concentrations were found in the DNG group (median 2.44 ng/ml, IQR: 1.12-4.70, P < 0.0001 versus non-DNG group, P = 0.048 versus non-endometriosis group). The treatment of primary cultured ESC with DNG (10(-5) M) suppressed MK mRNA production (P = 0.016), whereas MPA (P = 0.109) and NETA (P = 0.422) at same concentrations did not show a similar effect. LIMITATIONS, REASONS FOR CAUTION: The non-randomized design of the study. WIDER IMPLICATIONS OF THE FINDINGS: These findings could indicate a direct effect of DNG on endometriotic cells that could contribute to its effectiveness in the treatment of this disease. STUDY FUNDING/COMPETING INTERESTS: Funding was received from Swiss National Science Foundation (Grant No. 320030_140774). M.D.M. has received fees for speaking at scientific meetings from Bayer. The other authors have no conflicts of interest to declare.The authors state that the manufacturer of dienogest has in no way influenced the performance or outcomes of this study.

Serum but not follicular fluid cytokine levels are increased in stimulated versus natural cycle IVF: a multiplexed assay study.

Throughout follicular growth the number of immune cells increases, enhanced under stimulation with exogenous gonadotropins. This treatment, however, may adversely influence folliculogenesis and negatively affect oocyte quality through modifications in the follicular concentrations of cytokines released by these immune cells. We studied this hypothesis by systematically analysing the concentrations of cytokines present in the serum and follicular fluid at the time of follicular aspiration in conventional gonadotropin-stimulated (c-IVF) cycles in comparison with natural cycle IVF (NC-IVF) in which the follicles were naturally matured. Our study involved 37 NC-IVF and 39 c-IVF cycles including 13 women who underwent both therapies. Mean age was 35.3 ± 4.6 (SD) and 34.2 ± 3.7 years in the NC-IVF and c-IVF groups (ns). Thirteen cytokines were determined in matched serum and FF samples. Interleukin (IL)-4, TNF-α, RANTES, eotaxin and interferon-gamma-induced protein-10 concentrations were lower in FF than in serum. IL-6, -8, -10, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median levels in FF than in serum, indicating possible ovarian production. Most of these markers were also increased in concentration in the stimulated (c-IVF) than in the NC groups in the serum, but not in the follicular fluid. This finding can be attributed to the increased number of active follicles present after controlled ovarian stimulation. IL-8 was reduced in c-IVF cycles. Our study did not reveal differences in follicular fluid but in serum cytokine concentrations, suggesting that the follicular immune system might not be significantly affected by gonadotropin stimulation.

Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.

STUDY QUESTION: Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER: Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY: In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION: This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE: In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION: Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS: The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS: Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Regression of the inflammatory microenvironment of the peritoneal cavity in women with endometriosis by GnRHa treatment.

OBJECTIVE: To investigate the effect of gonadotropin-releasing hormone analogues (GnRHa) on the peritoneal fluid microenvironment in women with endometriosis. STUDY DESIGN: Peritoneal fluid was collected from 85 women with severe endometriosis (rAFS stage III and IV) during laparoscopic surgery during the proliferative phase. Prior to surgery clinical data were collected. The concentrations of specific markers for endometriosis in the peritoneal fluid were determined using an ELISA and a comparison between peritoneal fluid markers in women using GnRHa and no hormonal treatment was performed using a non-parametric Mann-Whitney U test. RESULTS: The study included peritoneal fluid from 39 patients who had been administered GnRHa (Zoladex(®)) in the three months prior to surgery and 46 from women with no hormonal treatment in this period. Concentrations of IL-8, PAPP-A, glycodelin-A and midkine were significantly reduced in the GnRHa treatment group compared to women receiving no hormonal treatment. RANTES, MCP-1, ENA-78, TNF-α, OPG, IP-10 and defensin showed no significant change between the two groups. CONCLUSIONS: GnRHa mediate a significant regression in the inflammatory nature of the peritoneal microenvironment in women with endometriosis.

Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies.

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

Androgen and gonadotropin patterns differ in HIV-1-infected men who develop lipoatrophy during antiretroviral therapy: a case-control study.

OBJECTIVES: We compared androgen and gonadotropin values in HIV-infected men who did and did not develop lipoatrophy on combination antiretroviral therapy (cART). METHODS: From a population of 136 treatment-naïve male Caucasians under successful zidovudine/lamivudine-based cART, the 10 patients developing lipoatrophy (cases) were compared with 87 randomly chosen controls. Plasma levels of free testosterone (fT), dehydroepiandrosterone (DHEA), follicle-stimulating hormone and luteinizing hormone (LH) were measured at baseline and after 2 years of cART. RESULTS: At baseline, 60% of the cases and 71% of the controls showed abnormally low fT values. LH levels were normal or low in 67 and 94% of the patients, respectively, indicating a disturbance of the hypothalamic-pituitary-gonadal axis. fT levels did not significantly change after 2 years of cART. Cases showed a significant increase in LH levels, while controls showed a significant increase in DHEA levels. In a multivariate logistic regression model, lipoatrophy was associated with higher baseline DHEA levels (P=0.04), an increase in LH levels during cART (P=0.001), a lower body mass index and greater age. CONCLUSIONS: Hypogonadism is present in the majority of HIV-infected patients. The development of cART-related lipoatrophy is associated with an increase in LH and a lack of increase in DHEA levels.

Serum leptin and C-reactive protein levels in the physiological spontaneous menstrual cycle in reproductive age women.

OBJECTIVE: Only a few studies have investigated variations of different markers for inflammatory processes during the physiological menstrual cycle. The results are conflicting, particularly concerning the correlation between the marker leptin and steroid hormones. The aim of the study was to investigate the inflammatory markers C-reactive protein (CRP) and leptin in the serum of healthy, normally ovulating women and to correlate these with each other and with the hormones of the gonadal axis. A cycle-dependence of the markers studied would imply an exact timing of the blood sampling for clinical needs. DESIGN: Observational study investigating the two inflammatory markers CRP and leptin in relation to the hormonal pattern of the gonadal axis during the normal cycle. METHODS: Ovulatory cycles of 36 healthy, young, normo-androgenic women, having a normal body mass index were evaluated. Serum concentrations of leptin and CRP, as well as of follicle-stimulating hormone, luteinising hormone, 17beta-oestradiol, progesterone, prolactin (PRL) and free testosterone were measured every 1-2 days during one full cycle. RESULTS: Serum levels of leptin and CRP behaved differently during ovulatory cycles, with higher concentrations for leptin only during certain phases. Significant correlations were found in the follicular phase between leptin and PRL and leptin and free testosterone. CONCLUSIONS: Leptin levels change during the menstrual cycle. Leptin levels are more stable on cycle days 1-5 than later in the cycle. For precise cycle-independent measurements, these fluctuations have to be taken into account. There is no similar cyclic pattern for CRP.

C-reactive protein production in term human placental tissue.

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Concentrations of leptin and C-reactive protein in serum and follicular fluid during assisted reproductive cycles.

BACKGROUND: There are only a few studies that have investigated inflammatory processes during ovarian hyperstimulation, with contradictory results especially concerning outcome. The aim of the study was to investigate the inflammatory markers C-reactive protein and leptin in serum and follicular fluid and to correlate these with the outcome. METHODS: One hundred and sixty-two gonadotrophin stimulated cycles were evaluated. Serum concentrations of leptin and C-reactive protein were measured at the initiation of stimulation, on the day of hCG administration or the day before, and on the day of oocyte retrieval. They were also determined in the follicular fluid. RESULTS: Serum leptin and C-reactive protein levels increased significantly during stimulation until the day of oocyte pick up, but following different patterns. After stimulation, they correlated with each other in serum and follicular fluid, but not with estradiol or progesterone concentration, embryo quality, or the pregnancy rate. CONCLUSIONS: Leptin and C-reactive protein levels change significantly during assisted reproductive treatment. In contrast to estradiol they are, however, not a marker of success.

Maternal serum levels of placental proteins after in vitro fertilisation and their implications for prenatal screening.

OBJECTIVES: To determine whether the serum levels of pregnancy-associated plasma protein A (PAPP-A), pregnancy-specific beta(1)-glycoprotein (SP1), placental lactogen (hPL) and human chorionic gonadotrophin (hCG) are different in pregnancies obtained after in vitro fertilisation (IVF) and embryo transfer (ET) in comparison to spontaneous pregnancies. Assessment of the need to establish normal medians for biochemical trisomy screening in IVF pregnancies. METHODS: The population comprised 96 IVF-ET pregnancies, of which 79 came from fresh gonadotrophin-stimulated cycles and 17 from embryo transfers without gonadotrophin stimulation (natural cycle IVF, frozen embryo transfers), and 156 spontaneous pregnancies. A single blood sample was obtained between 7 + 0 and 16 + 3 weeks. PAPP-A, SP1, hPL and hCG were quantified and the levels compared between gonadotrophin-stimulated IVF, steroid-only- or non-stimulated IVF, and controls with respect to gestational age using non-parametric statistical analysis. RESULTS: PAPP-A and hPL levels were reduced after stimulated IVF in early gestation (before 10 pregnancy weeks); SP1 followed the same trend without reaching statistical significance. hCG tended to be increased after IVF treatment including non-gonadotrophin-stimulation cycles, and also beyond 10 pregnancy weeks. CONCLUSION: Reduced PAPP-A with increased hCG yields an increased risk in screening for foetal trisomy 21. We confirm recently published observations but do not recommend the establishment of normal medians for IVF pregnancies since the extent of the deviations is varying according to the different stimulation protocols and dosages of gonadotrophins used.

First-trimester maternal serum PAPP-A, SP1 and M-CSF levels in normal and trisomic twin pregnancies.

OBJECTIVE: To study PAPP-A and SP1 for biochemical trisomy screening in twin pregnancies and to investigate the role of maternal and placental compartments in marker production by comparing the levels of the decidual cytokine M-CSF with the PAPP-A and SP1 from the placenta. METHODS: Thirteen twin pregnancies with at least one chromosomally abnormal fetus were compared with 68 normal twin pregnancies. Sera were obtained between 11 + 3 and 13 + 6 weeks of gestation, and PAPP-A, SP1 and M-CSF levels were determined by immunoassay. These concentrations were also compared with gestation-matched groups of 18 singleton normal pregnancies and 18 singleton Down syndrome pregnancies. RESULTS: PAPP-A and SP1, but not M-CSF, levels were higher in normal twin pregnancy than in normal singleton pregnancy. SP1 levels, but not PAPP-A, correlated to M-CSF. PAPP-A, but not SP1, levels were reduced in abnormal twin pregnancies, with an increasing effect according to the number of affected fetuses, and were more pronounced in pregnancies with trisomy 18 or 13 than in trisomy 21 fetuses. M-CSF was inconsistent, with a trend towards increased levels in trisomy 21. CONCLUSION: PAPP-A remains the best biochemical screening marker for fetal trisomies 21, 18 or 13, in singleton as well as in twin pregnancy. In contrast to SP1, its site of production is not likely to be restricted to the placenta. The role of the (maternally produced) M-CSF remains to be further investigated.

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.

Production of protein hormones by cultured trophoblast cells isolated from term and early placentae.

PROBLEM: To compare the capacity of de novo hormone synthesis by cultured trophoblast cells isolated from early and term placenta as cytotrophoblast, and to determine the ability of these cells to proliferate in culture. METHOD OF STUDY: Cytotrophoblast cells were isolated from term (TP, 38-42 weeks) and early placentae (EP, 8-13 weeks) by enzymatic digestion and subsequent purification on a percoll gradient. The net synthesis of the hormones human placental lactogen (hPL) and human chorionic gonadotropin (hCG) was determined as the release during culture + cell content after culture - cell content before culture. Proliferation was determined using a dedicated colorimetric reagent (CellTiter 96). RESULTS: Using a percoll gradient we were able to isolate three cell bands with densities of 1.051, 1.058, and 1.063 g/mL, which were predominantly cytotrophoblast cells as shown by immunocytochemical analysis. The cytotrophoblast cells with the highest density (1.063 g/mL) were used because they were found to release the highest amount of hormones and have shown the lowest rate of cell death after 6 days in culture. Both hCG and hPL showed different patterns of release during the first 2-3 days of culture between TP and EP. While the release by EP cytotrophoblast cells continued during 6 days of culture (n = 4), the concentrations for TP cytotrophoblast (n = 4) reached a plateau between 4 and 6 days. Net de novo synthesis calculated for 3 x 10(4) TP trophoblast cells cultured for 6 days (mean +/- SD, n = 4) was 8.65 +/- 9.05 mU for hCG and 0.95 +/- 0.45 ng for hPL. For EP, it was 395.5 +/- 265.5 mU for hCG and 148.8 +/- 84.2 ng for hPL. Net synthesis of hCG was > 10-fold (TP) and > 70-fold (EP) higher than the initial cell content. While at term, hPL synthesis was only a fraction of the initial cell content, production by EP cytotrophoblast was 106 times the initial cell content. The extent of cell death after 6 days in culture was significantly (P < 0.02) higher for term (30-40%) than for early trophoblast (10-20%). Using a proliferation detection agent during the first 3 days of culture with first trimester cytotrophoblast cells, we did not find any changes in the proliferative activity. CONCLUSIONS: There are differences in the functional activity between trophoblast cells obtained from first and third trimester. The in vitro findings are difficult to reconcile with the different patterns of plasma concentrations of the two hormones observed in vitro during the course of pregnancy.

Aging, serum estradiol levels, and pregnancy differentially affect vascular reactivity of the rat uterine artery.

OBJECTIVES: To investigate the effects of aging, ovarian ablation, and pregnancy on vascular reactivity of the rat uterine artery. METHODS: Segments of uterine artery from 3-month-old pregnant and nonpregnant Wistar rats and from aged and ovariectomized animals, both 9 months of age, were exposed in vitro to vasoactive mediators. Absolute contractile force as well as endothelium-dependent and -independent vascular reactivity were determined. Isometric tension was recorded using a modified Mulvany myograph system. Results were compared with analysis of variance and Bonferroni-Dunn post hoc analysis and correlated with serum estradiol levels. RESULTS: Aging up to 9 months decreased absolute tension of uterine arteries in vitro elicited by KCl (P < .0001), while not affecting receptor-operated responses to norepinephrine, endothelin-1, and angiotensin II. After ovarian ablation maximal contraction to norepinephrine was selectively reduced in the aged animal (P = .0053). Pregnancy increased absolute tension to KCl (P < .0001), norepinephrine (P < .008), and endothelin-1 (P = .0003), whereas relative contractile force (percentage of KCl) induced by norepinephrine and endothelin-1 remained unchanged and that induced by angiotension II decreased (P = .0001) in pregnant animals. In addition, pregnancy increased sensitivity to the endothelium-dependent vasodilator acetylcholine (P = .0022) but decreased that to the endothelium-independent vasodilator sodium nitroprusside (P = .0062). Endothelium-dependent relaxation correlated with serum estrogen levels remained unchanged in 9-month-old Wistar rats, which physiologically exhibited high serum estrogen concentrations but was impaired with regard to both maximum relaxation (P < .0001) and sensitivity in aged rats (P = .0007) after ovariectomy. CONCLUSIONS: Vascular contractility is impaired in the uterine artery of the aged rat as evidenced by reduced responses to KCl, whereas responses to receptor-operated agonists remain unchanged. Functional ovaries are essential to preserve endothelium-dependent relaxation in aging animals. During pregnancy, contractile machinery and endothelium-dependent relaxation are enhanced. In contrast, contractions to angiotensin II and endothelium-independent relaxation to sodium nitroprusside are reduced in late pregnancy. These changes in reactivity of the uterine artery may be important for the regulation of blood flow in the uterus according to physiologic needs.

Decrease of serum endothelin levels with postmenopausal hormone replacement therapy or tibolone.

Endothelin is the most potent vasoconstrictor peptide known to date. Hormone replacement therapy (HRT) with estrogen reduces plasma endothelin levels. We measured endothelin in 51 postmenopausal patients before and during HRT. Patients were randomly allocated to receive either oral tibolone, oral or transdermal 17 beta-estradiol. A group of comparable volunteers served as controls. After 24 months, endothelin levels decreased in all treatment groups: tibolone, 18.2%; oral 23.1%; transdermal, 20.8%. Endothelin levels increased in the controls by 36.6% (p < 0.01). Tibolone decreases endothelin levels to a similar degree as conventional estrogen-progestogen-replacement therapy. These data provide another potential mechanism supporting the cardioprotective effects of tibolone.