Immunological factors in endometriosis-associated reproductive failure: studies in fertile and infertile women with and without endometriosis

Immunopathophysiological mechanisms in endometriosis-associated reproductive failure were studied in appropriate populations: infertile and fertile women with and without endometriosis. The incidence of sera positive for any of the autoantibodies tested among infertile women with endometriosis (n = 25) was similar to that observed in the three control groups [unexplained infertility patients (n = 25) and fertile women with (n = 10) and without (n = 25) endometriosis]. The mean volume of peritoneal fluid was significantly elevated in women with endometriosis (both fertile and infertile) as compared with patients without endometriosis (fertile or infertile). The concentration of peritoneal fluid leukocytes and the percentage of cells positive for macrophage markers were significantly increased and the percentage of T lymphocytes significantly decreased in infertile women with endometriosis but not in patients with unexplained infertility and fertile women with endometriosis, as compared with fertile controls without endometriosis. Macrophages from infertile patients with endometriosis had higher sperm phagocytosis than did those from infertile women without endometriosis or fertile subjects with or without endometriosis. Incidences of serum and peritoneal fluid samples embryotoxic to the in-vitro development of 2-cell mouse embryos were significantly higher in infertile patients with endometriosis than in unexplained infertility patients and fertile women with or without endometriosis. It is concluded that immunological mechanisms of endometriosis-associated infertility exist but that these peritoneal immunological factors in infertile women with endometriosis are related to their subfertility rather than to the presence of ectopic endometrial implants. This is supported by the lack of immunological abnormalities observed among fertile women with endometriosis. These immunological abnormalities are lacking in patients with unexplained infertility.      

Differences in time to natural conception between women with unexplained infertility and infertile women with minor endometriosis

BACKGROUND: Opinion remains divided as to whether finding endometriotic lesions in the absence of adhesions has an adverse effect on the likelihood of conception. METHODS: This was a retrospective study of 192 fully investigated infertile couples, followed up for up to 3 years following laparoscopy. Women studied were ovulating, <40 old years and their partners had normal sperm parameters. All 117 women with unexplained infertility and 75 with minimal/mild endometriosis without adhesive disease were managed conservatively. RESULTS: Women with endometriosis were found to have a lower probability of pregnancy compared with women with unexplained infertility (36% versus 55%; P<0.05). Other factors adversely associated with pregnancy were primary infertility, smoking and longer duration (>3 years) of infertility. However, the effects of duration of infertility and primary infertility were not observed to be statistically significant for women with endometriosis. CONCLUSIONS: The findings, although undertaken in a select population undergoing laparoscopy, suggest the likelihood of pregnancy is reduced in infertile women with minimal/mild endometriosis compared with those infertile women with a normal pelvis. Duration of infertility and a previous history of pregnancy are important in predicting the likelihood of pregnancy in women with no obvious cause for their infertility (unexplained), whilst the relationship may be more complex in women with minor endometriosis     

Infertility: Infertility evaluation in fertile women: a model for assessing the efficacy of infertility testing

A standard infertility evaluation consists of a semen analysis,hysterosalpingogram, post-coital test, endometrial biopsy andlaparoscopy. Although these tests are well grounded in clinicalexperience, information on their ability to discriminate betweenfertile and infertile couples is limited. In this study, weperformed standard infertility tests plus two others-sperm antibodiesand cervical culture for Mycoplasma hominis and Ureaplasma urealyticum–onfertile and infertile couples. Women in the fertile group wereselected from those who had delivered a child within the previous2 years and who were scheduled for a laparoscopic tubal ligation.Women in the infertile group were selected from those presentingfor an infertility evaluation (mean duration of infertility4.2 years), and they were matched by age (±3 years) andrace with fertile subjects. Subjects were recruited from bothprivate and clinic patients. A total of 64 couples (32 matchedpairs) completed the evaluation. At least one ’abnormal‘infertility test was found in 69% of fertile and 84% of infertilecouples. With the exception of tubal damage and endometriosis,which as expected were more common in infertile couples, nosignificant differences between groups for remaining infertilityfactors could be demonstrated. Despite the small size of thecurrent study, these results confirm the feasibility and importanceof comparisons of the prevalence of infertility factors in fertileand infertile couples.     

Leukaemia inhibitory factor and interleukin 11 levels in uterine flushings of infertile patients with endometriosis

BACKGROUND: Exact aetiology of infertility in stage I/II endometriosis patients is not known. Interleukin 11 (IL-11) and leukaemia-inhibitory factor (LIF) are factors associated with implantation window in human eutopic endometrium. We decided to test whether there is an altered secretion of these factors, which could explain receptivity defect in patients with minimal endometriosis. METHODS: Uterine flushing and endometrial samples were collected 7-9 days after ovulation (implantation window) from infertile patients with stage I/II endometriosis (n = 14) and fertile, endometriosis-free controls (n = 21). IL-11 and LIF were assessed in uterine flushings in eutopic endometria in all patients by enzyme-linked immunosorbent assay (ELISA). In eutopic endometrium, semiquantitative RT-PCR was performed for LIF and IL-11 mRNA expressions. RESULTS: No statistically significant differences were found in uterine flushing in women with and without endometriosis with regard to IL-11 levels (0.0 pg/ml versus 0.0 pg/ml) and LIF (25.53 pg/ml versus 36.26 pg/ml). These results were confirmed by the results of RT-PCR, where there were also no differences between studied groups. CONCLUSIONS: There is no receptivity defect with regard to LIF and IL-11 secretions by eutopic endometrium in infertile women with endometriosis.     

Transferrin Receptor (CD71) Expression in Peritoneal Macrophages from Fertile and Infertile Women With and Without Endometriosis

PROBLEM: Hyperactivated macrophages are implicated in the pathophysiology of endometriosis-associated infertility. This study investigates transferrin receptor expression (CD71) as a marker of hyperactivity in peritoneal macrophages of infertile patients with minimal to mild endometriosis (group 1, n = 25). METHOD OF STUDY: Expression of the activation antigen CD71 on peritoneal fluid macrophages was determined by a specific monoclonal anti-CD71 antibody using indirect immunofluorescence technique and was analyzed by flow cytometry. Three different control groups of women were used: women with unexplained infertility (group 2, n = 25), fertile women with endometriosis (group 3, n =10), and fertile women without endometriosis (group 4, n = 25). RESULTS: The percentage of CD71 positive cells was significantly increased in infertile women with endometriosis as compared with the three control groups. There were no differences among groups 2, 3, and 4 with respect to the percentage of CD71 positive macrophages. CONCLUSIONS: Our results favor the concept that hyperactivated macrophages play a role in the pathophysiology of endometriosis-associated subfertility, a feature which is lacking in patients with unexplained infertility.     

Serum prolactin and thyrotropin responses to thyrotropin-releasing hormone in men with alcoholic cirrhosis

The serum concentrations of prolactin (PRL) and thyrotropin (TSH) in 12 males with alcoholic cirrhosis during basal condition and after stimulation with thyrotropin-releasing hormone (TRH) were compared with the concentrations in ten thiazide-treated hypertensive and nine normal men. The basal as well as the TRH-stimulated increase in serum PRL was significantly elevated in the cirrhotic males, while the increase in serum TSH was unchanged, compared with hypertensive and normal men. No correlation between clinical or laboratory parameters and serum PRL was found. Serum estradiol was equal in cirrhotic and control subjects and no correlation was found between serum PRL and serum estradiol. These findings favour the concept that raised serum PRL in cirrhotic patients might be caused by a diminished dopaminergic neurotransmission.     

Tubal damage in infertile women: prediction using chlamydia serology

BACKGROUND: The study explores the relationship between serum chlamydia antibody titres (CATs) and detection of tubal damage in infertile women. METHODS: The tubal status and pelvic findings in 1006 women undergoing laparoscopy for infertility were related to CAT, which was measured using the whole-cell inclusion immunofluorescence test. RESULTS: A negative correlation between CAT and age was noted. A linear trend between serum CAT and the likelihood of tubal damage, including severe damage, was observed (P < 0.001). Titres in women with tubal damage (median 1:1024; range <1:64-1:4096) were significantly (P < 0.001) higher than in women with endometriosis alone (median <1:64; range <1:64-1:512) or those with a normal pelvis (median <1:64; range <1:64-1:1024). Women with positive titres were more likely to have pelvic adhesions than tubal occlusion unless titres were very high, when tubal damage was likely to be more severe. CONCLUSIONS: CATs are of predictive value in the detection of tubal damage and are quantitatively related to the severity of damage. For practical clinical purposes, Chlamydia serology is useful mainly as a screening test for the likelihood of tubal damage in infertile women and may facilitate decisions on which women should proceed with further investigations without delay.     

Pituitary--ovarian dysfunction as a cause for endometriosis-associated and unexplained infertility

This study examines circulating and follicular hormone concentrationsand fertilization of oocytes in cycles totally unperturbed byexogenous gonadotrophins in 10 women (25 cycles) with untreatedminimal—mild endometriosis and nine women (23 cycles)with prolonged unexplained infertility compared with 16 women(50 cycles) with tubal damage as functional controls. Endometriosiswas associated with a significantly longer follicular phase(median 15,12,13 days respectively) and reduced oestrogen secretion(median index area under the curve 3063, 3842, 3805 units respectively)compared with controls. Both endometriosis and unexplained infertilityhad significantly reduced serum luteinizing hormone (LH) surges[median peak serum (LH) 43, 39, 55 IU/1 respectively and medianarea under the curve 661, 687, 823 units respectively] and reducedLH concentrations in follicular fluid (median 19.6, 10.6, 9.2IU/1 respectively). These findings suggest that infertilityassociated with minor endometriosis and of apparently unexplainedaetiology share a common pathophysiology in impaired LH surgesecretion. Whether that represents a primary pituitary disorderor is secondary to a defective ovarian signal is discussed.     

Analysis of CTLA4 gene variant in infertile Brazilian women with and without endometriosis

An autoimmune aetiology has been suggested for endometriosis mostly on the basis of an increased prevalence of autoimmune diseases in affected women. Cytotoxic T lymphocyte antigen (CTLA) 4 gene is recognized as a primary determinant for autoimmunity, since specific polymorphisms have been associated with predisposition to most autoimmune disorders. Thus, the objective of the study was to evaluate CTLA4 polymorphism (+49A/G) in a group of infertile women with and without endometriosis and controls. Case-control study comprising 244 infertile women (177 with endometriosis and 67 without endometriosis) and 172 fertile women as controls. CTLA4 polymorphism was identified by qPCR. The results were analysed statistically and a P-value <0.05 was considered significant. We found relatively similar CTLA4 polymorphisms genotype frequencies in women with and without endometriosis and controls (P=0.158 and P=0.262, respectively). When the patients with minimal/mild endometriosis and moderate/severe endometriosis were studied separately, no difference was also found related to controls (P=0.560 and P=0.11, respectively). The data suggest that the CTLA4 polymorphism is not associated with endometriosis and/or infertility in Brazilian women.     

Investigation of the infertile couple: should diagnostic laparoscopy be performed after normal hysterosalpingography in treating infertility suspected to be of unknown origin?

Traditionally, a diagnosis of unexplained infertility is established only when all standard clinical investigations yield normal results. When tubal patency has been established by hysterosalpingography (HSG), laparoscopy has been suggested as a mandatory step to preclude the existence of peritubal adhesions and endometriosis as causes of infertility. In women without a previous history suggestive of tubal disease and who have a normal HSG, it was demonstrated that the probability of clinically relevant tubal disease or endometriosis is very low and that laparoscopy does not seem justified or cost effective. In the minority of these cases, laparoscopy might reveal minimal or mild endometriosis or peritubal adhesions. In these cases, either surgery or medical treatment has not been proven to improve fecundity. With the current success rates of assisted reproductive technologies (ART) and the relatively low contribution of diagnostic laparoscopy to the decision-making process of treating patients with a normal HSG, we suggest that laparoscopy should be omitted in couples suspected of having unexplained infertility. These patients should be treated by 3-6 cycles of combined gonadotrophins and intrauterine insemination, and if unsuccessful switched to ART.     

Natural cycle IVF in unexplained, endometriosis-associated and tubal factor infertility.

BACKGROUND: To elucidate possible differences between unexplained and minimal peritoneal endometriosis-associated infertility, we studied their outcome in natural cycle IVF (NIVF). METHODS: A prospective cohort study was carried out on unexplained (33 couples), minimal peritoneal endometriosis-associated (30 couples) and tubal factor (24 couples) infertility in 223 NIVF cycles, using human chorionic gonadotrophin (HCG) for ovulation induction. RESULTS: During the first NIVF attempt, follicular and luteal phase oestradiol, FSH, LH and progesterone concentrations, as well as endometrial thickness and follicular diameter were similar among the three groups. Periovulatory follicular growth monitored from day of HCG administration to oocyte aspiration was significantly lowered in unexplained infertility compared with minimal endometriosis-associated and tubal factor infertility. The fertilization rate, clinical pregnancy rate per initiated cycle, per successful oocyte retrieval and per embryo transfer, in minimal endometriosis (80.0, 10.4, 16.0 and 23.5% respectively) were similar to that in tubal factor infertility patients (68.6, 5.8, 11.4 and 16.0%) but significantly higher (P < 0.05) than that of the unexplained infertility group (62.2, 2.6, 5.4 and 8.7%). CONCLUSIONS: The significant reduction in follicular periovulatory growth, fertilization and pregnancy rates in unexplained infertility compared with minimal peritoneal endometriosis patients may be explained by sub-optimal follicular development with possibly reduced oocyte quality, intrinsic embryo quality factors or by impaired implantation. From a clinical point of view, NIVF is less suited to unexplained infertility treatment, but might represent an interesting treatment option for minimal peritoneal endometriosis-associated infertility.     

Acute dopaminergic blockade augments the naloxone-induced LH rise in estrogen-treated postmenopausal women

Objectives: To assess the effect of estrogen replacement on the simultaneous blockade of the dopaminergic (DA) and opioidergic neural control of hypothalamic-gonadotropic function in postmenopausal women. Methods: Twenty healthy postmenopausal women, 48–55 years old were randomly assigned to receive either a 4-h naloxone infusion at 2 mg/h (group 1, n = 7) or a 10 mg i.v. bolus of metoclopramide (group 2, n = 7) or both drugs, simultaneously (group 3, n = 6) before and after 3 weeks of transdermal estradiol (100 μg/day). Blood samples were obtained at 30-min intervals during 4 h and duplicate determinations of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂) and prolactin (PRL) were performed in all samples. Results: In group 1 only a mild but significant LH rise after but not before estrogen replacement was seen. In group 2 PRL had a greater rise after than before estrogen therapy, without other hormonal changes. In group 3 a greater rise in PRL occurred after than before estrogen administration and serum LH had a sustained rise throughout the test only after estrogen replacement (greater than in group 1). No FSH changes were observed. The after-estradiol PRL response was nearly similar in groups 2 and 3. Conclusions: Our results indicate that in the untreated postmenopausal women, the dopaminergic system has little and the opioidergic system has no significant input in the control of gonadotropin or prolactin release. However, following estrogen replacement, opioids are involved in the inhibition of LH release and stimulating PRL release, while the dopaminergic system acts to inhibit PRL release and modulates LH release or inhibition, depending on the levels of circulating estrogens.     

Autoantibodies and antisperm antibodies in sera and follicular fluids of infertile patients; relation to reproductive outcome after in-vitro fertilization

Immune reactions have effects at various concentrations in thereproductive process and autoantibodies may have an impact onfertility and the outcome of assisted conception. We measuredthe prevalence of and relation between antibodies to smoothmuscle, nuclear, phospholipid and sperm antigens, and concentrationsof immunoglobulins G, M and A and complement components C3 andC4, in the sera and follicular fluids of women with unexplainedinfertility (n = 30), endometriosis (n = 20), tubal infertility(n = 50) and the sera of 20 normal non-pregnant women. We assessedfertilization and successful pregnancy rates in relation toantibody status of infertile women after in vitro fertilization.All antibodies had a higher prevalence in infertile women comparedwith controls and this was significant for smooth muscle antibodyin endometriosis (P < 0.05); anticardiolipin antibody intubal infertility P < 0.05); and antisperm antibody in alltypes of infertility (P < 0.001). There was no relation betweenpresence of specific antibodies in serum or between serum andfollicular fluids. Total biochemical pregnancy rate was higherwith endometriosis (P = 0.05) but clinical pregnancy and livebirth rates did not differ between groups or in relation toantibody status. Significant differences in immunoglobulin andcomplement components occurred in women with and without successfulbiochemical pregnancy.     

Endometrial development in the luteal phase of women with various types of infertility: comparison with women of normal fertility

In this study, the prevalence of retarded endometrial development in the luteal phase of an infertile population (20/142 = 14%) was found to be significantly higher (P less than 0.05) than in that of a control, fertile population (3/68 = 4.4%). However, when the infertile subjects were subdivided into groups of different aetiology, it was found that women with endometriosis and unexplained infertility had a significantly higher prevalence of retarded endometrium (6/21 = 29%, 10/48 = 21% respectively; P less than 0.01 in both cases) than women with normal fertility; whereas the prevalence in women with tubal or male infertility (1/34 = 2.9% and 3/39 = 7.7%, respectively) was not significantly different from that in the fertile subjects. There were no significant differences in the progesterone profiles of the four groups of infertile subjects and the group of fertile subjects. Of the 15 cases of retarded endometrial development with known progesterone profile, two were associated with subnormal progesterone whereas the remaining 13 were associated with normal progesterone, the latter suggesting an abnormal response of the endometrium to a normal amount of progesterone. The findings suggest that further morphological study of the endometrium should be aimed at subjects whose infertility is unexplained or associated with endometriosis, in whom the prevalence of abnormal endometrium is increased.     

Endometriosis and infertility: our experience over five years

During a 5-year period, we diagnosed endometriosis by laparoscopyin 163 infertile patients (7.9% of the Infertile population).Seventy-one women presented with minimal endometrlosis, 32 womena mild form, 38 women a moderate form and 22 women a severeform. Only 40 patients (24.5%) were found to have endometriosisas the only abnormality in a complete fertility work-up. Onehundred and twenty patients were treated with Danazol (600 mgdaily for 4–6 months) and 33 of them conceived (27.5%).Twenty-eight patients received conservative surgical treatmentassociated with Danazol therapy and 11 of them conceived (39.2%).In the 15 patients with minimal endometriosis, who had no therapybut expectant management, there were two conceptions withinone year (13.3%). The anticipation of minimal disease In patientswith endometriosis as the only abnormality produced about thesame results as achieved by active treatment (pregnancy rateof 33.3% after expectant management and 36.3% after treatment).These results suggest that minimal endometriosis can be anticipatedonly when there are no co-existing factors of infertility.     

Long-term fertility prognosis following selective salpingography and tubal catheterization in women with proximal tubal blockage

BACKGROUND: The possibility of conception following selective salpingography and tubal catheterization is believed to decline sharply a few months after the procedure. This observation may be due to the relatively small number of patients and short follow-up of previous studies. Furthermore, couples with other causes of infertility apart from proximal tubal blockage have usually been excluded. METHODS: Survival analysis of conceptions of 218 consecutive infertile women with proximal tubal blockage who underwent selective salpingography and tubal catheterization was performed. There were no exclusion criteria. Follow-up ranged from 16 to 56 months. RESULTS: A total of 47.2% of spontaneous conceptions and 43.2% of all conceptions, apart from those achieved by IVF or ICSI treatments, occurred after the first 12 months following selective salpingography and tubal catheterization. The decline in the possibility of pregnancy during the study period (conception hazard rate) was only minimal. CONCLUSIONS: In a population of infertile women with proximal tubal blockage, a significant proportion of conceptions occur after the first 12 months following selective salpingography and tubal catheterization. The presence of any additional causes of infertility in the couple should not be regarded as an absolute contraindication to the procedure.     

Oxidative stress and endometriosis

BACKGROUND: Little is known about the aetiology of endometriosis; however, in the presence of oxidative stress, reactive oxygen species might increase growth and adhesion of endometrial cells in the peritoneal cavity, leading to endometriosis and infertility. Within a study investigating persistent organic compounds and endometriosis, the authors evaluated the association between oxidative stress and endometriosis. METHODS: Women aged 18-40 years who were undergoing laparoscopy were contacted to participate in the study (n = 100); 84 were eligible and agreed to be interviewed; 78 provided blood specimens. Four markers of oxidative stress and antioxidant status were measured in serum for 61 women. Multiple imputation of missing data was used to generate values for the missing oxidative stress data. RESULTS: Thirty-two women had visually confirmed endometriosis at laparoscopy while 52 did not, including 22 undergoing tubal ligation and 30 with idiopathic infertility. There was a weak association between thiobarbituric acid-reactive substances (nmol/ml) and endometriosis, after adjusting for age, body mass index, current smoking, hormone use in the past 12 months, gravidity, serum vitamin E, serum estradiol, and total serum lipids (beta = 1.18; 95% CI-0.04, 2.39). CONCLUSIONS: These results suggest that oxidative stress might play a role in the development and progression of endometriosis, which should be evaluated in larger studies.     

Embryotoxicity of peritoneal fluid in women with endometriosis. Its relation with cytokines and lymphocyte populations

BACKGROUND: The goals of the present work were to study the embryotoxic effects of peritoneal fluid (PF) in women with or without endometriosis, and to relate any embryotoxicity to the severity of endometriosis, infertility or achievement of pregnancy, cytokine concentrations and lymphocyte populations. METHODS: Sixty-six consecutive women of reproductive age, 54 with endometriosis (21 infertile) and 12 infertile without endometriosis, and another 12 fertile women as control group, were included in this study. They all underwent laparoscopy or laparotomy in the second half of the cycle, and PF was collected from the pouch of Douglas. The embryotoxicity of the PF was assessed by means of a mouse embryo assay, and expressed as the number of embryos that did not reach blastocyst stage. Cytokines and lymphocyte populations present in PF were also studied and correlated with embryotoxicity. RESULTS: PF embryotoxicity was increased in women with endometriosis, but there was little correlation with the severity of the disease. However, although a clear relationship to the presence of infertility was not found, embryotoxicity appeared to be lower in those infertile patients with endometriosis who later became pregnant. We found a significant increase in embryotoxicity in the presence of high cytokine concentrations, especially with interleukin-6, and less so with interleukin-8 (P < 0.05). No good correlation was observed with lymphocyte populations, but CD56 (NK) cells were significantly increased in the PF of women with endometriosis. In general, the correlations for embryotoxicity were better when PF was diluted at 20% (91.4 +/- 17 versus 68.1 +/- 31, P < 0.01). CONCLUSIONS: These results suggest that alteration in the production of cytokines in the PF, especially IL-6, besides contributing to the endometriosis and its evolution, probably increases embryotoxicity. However, no correlation was found between the latter and associated infertility.     

Tubal infertility and silent chlamydial salpingitis

Antibodies to Chlamydia trachomatis (serum IgG antibodies witha titre of at least 32) were detected in 141 (86.0%) of 164infertile women with tubal infertility (TF group) and in 20(28.6%) in 70 infertile women with normal tubes (NTF group).The difference was highly significant (P < 0.001), as wasthe difference in geometric mean titre of antibody-positivecases, 181 and 87, respectively. Pregnant, agematched womenwere used as controls. Significantly fewer of these women (P< 0.001) had antibodies, when compared with the TF group,60 (36.6%) of 164, but not when the comparison was made withthe NTF group, 31 (44.3%) of 70. There was a history of salpingitisin 64 (39.0%) of the 164 infertile women with damaged tubes.Neither the frequency nor the geometric mean titre of chlamydialantibodies differed between tubal factor infertility patientswith and without a history of salpingitis. Previous chlamydialinfection, reflected by serological markers, is strongly associatedwith tubal damage leading to tubal infertility. A large proportionof these cases run a silent course, since a majority of theantibody-positive patients with tubal infertility have neverhad salpingitis     

Hybridization studies of cultured human pituitary prl and gh producing adenoma cells: Effects of thyrotropin-releasing hormone,somatostatin, and phorbol ester

The effects of the hypothalamic hormones, thyrotropin-releasing hormone (TRH), and somatostatin (SRIH), and of phorbol 12-myristate 13-acetate (PMA) on PRL and GH secretion and messenger RNA (mRNA) levels were analyzed in 10 GH and/or PRL producing adenomas after culturing the tumor cells in the presence of these secretagogues for 7 days. The expression of chromogranin A and B mRNAs was also examined. All four of the clinically diagnosed GH adenomas expressed or secreted both GH and PRL while four of six clinically diagnosed prolactinomas produced or secreted both PRL and GH. Prolactinomas had less than 10% of tumor cells expressing chromogranin A mRNA while more than 40% of the adenoma cells expressed chromogranin B mRNA. TRH stimulated PRL secretion and increased PRL mRNA levels while SRIH decreased GH secretion and mRNA expression in some cases. Unexpectedly, PMA stimulated PRL mRNA levels four- to sevenfold above control levels in two adenomas and generally stimulated chromogranin A and B mRNA expression but not GH mRNA, as determined by Northern hybridization and in situ hybridization analyses. These results indicate that cultured prolactinoma cells express significantly more chromogranin B mRNA than chromogranin A mRNA, and that PMA increases PRL mRNA expression in some prolactinomas, although the effect of PMA on various adenomas reflects the heterogeneity of these tumors with respect to protein kinase C stimulation.