L. J. Hipkin

The role of a single progesterone measurement in the diagnosis of early pregnancy failure and the prognosis of fetal viability

Objective To assess the role of a single maternal serum progesterone measurement in the immediate diagnosis of early pregnancy failure and in the long term prognosis of fetal viability.

PLASMA OESTRIOL AND HUMAN PLACENTAL LACTOGEN MEASUREMENTS IN PATIENTS WITH HIGH RISK PREGNANCIES

Maternal plasma oestriol and human placental lactogen (HPL) were measured serially in 383 at‐risk pregnancies. Eighty‐five infants were growth retarded and 122 developed fetal distress or neonatal asphyxia. Of the infants whose mothers had either abnormal plasma oestriol or HPL levels, 58 per cent were growth retarded, while 65 and 73 per cent in each group respectively developed fetal distress. The incidence of fetal complications when both plasma oestriol and HPL were abnormal was consistently greater than 66 per cent.

Plasma progesterone levels as an index of ovulation

Summary. Plasma progesterone levels were measured in three groups of untreated women. (1) Nine women with follicle rupture, proved by laparotomy or laparoscopy, had values of ≥40 nmol/l on days 18–24 (days −10—5 from the next period); thus a value of <40 nmol/1 should not be taken as evidence of ovulation. (2) Nineteen healthy women with normal menstrual histories had hormone assays on alternate days during one cycle. In five of them all values were <38 nmol/1. (3) Forty women had a single progesterone assay on days 20–29 of a conceptual cycle. Eight of them had a level <40 nmol/l. Ovulation had certainly occurred in all of them, but it is difficult to assess whether the sample timing was optimal since there was no following menstrual period. Progesterone levels in drug‐induced conceptual cycles were in general higher than those in spontaneous pregnancy cycles. Women with luteinization of the unruptured follicle frequently had values of >40 nmol/l. Conversely, a secretory endometrium was not uncommon in cycles with values of <38 nmol/l.

Effects of insulin-like growth factor-I on growth hormone and prolactin secretion and cell proliferation of human somatotrophinomas and prolactinomas in vitro

OBJECTIVE IGF‐I inhibits GH secretion from normal and some tumorous pituitary tissue, and has been shown to be mitogenic for gonadotrophinoma cells in vitro. It is not known whether IGF‐l affects somatotrophinoma cellular proliferation or the secretion of other hormones, such as PRL and α‐subunit, which are often co‐secreted by these tumours. We have therefore examined the effects of IGF‐l on proliferation and hormonal secretion of human somatotrophinomas and prolactinomas in vitro.

HYPOTONIC LYSIS OF RED BLOOD CELL CONTAMINATION FROM HUMAN ANTERIOR PITUITARY ADENOMA CELL PREPARATIONS

Dear Editor: The purpose of this letter is to inform fellow scientists of a simple and reliable method for the removal of red blood cell (RBC) contamination from human anterior pituitary cell preparations, facilitating pituitary cell attachment for dynamic studies in vitro. Contaminating RBC attach rapidly and avidly to cell culture surfaces and may prevent the attachment of anterior pituitary adenoma cells in vitro (4). It is unclear whether the physical presence of RBCs reduces the surface area available for cell attachment or subsequent RBC lysis liberates toxic products, such as iron, which has been shown to inhibit rat pituitary clonal cell growth in vitro (6). However, red blood cell removal may damage other cell types with percoll being associated with membrane instability in the purification of canine neutrophils (3). Nine human pituitary adenomas; three somatotrophinomas, one prolactinoma, and five clinically nonfunctional adenomas (NF adenomas) were dispersed to single cells using a combination of a 200

The effect of clomiphene citrate treatment on cervical mucus and plasma estradiol and progesterone levels

OBJECTIVES To study the relationship between cervical mucus (CM) quality, postcoital test (PCT) results and plasma estradiol (E2) in clomiphene citrate (CC)-treated women. A subsidiary aim was to study the relationship between CM quality and plasma progesterone (P). DESIGN Untreated women were compared with oligo-ovulatory patients given CC. SETTING Infertility Clinic, Fazakerley Hospital, United Kingdom. PATIENTS, PARTICIPANTS Fifty-one untreated patients and 31 women given CC. INTERVENTIONS The treated women were given 50 mg/d CC from days 2 to 6 of their cycle. MAIN OUTCOME MEASURES A CM assessment, a PCT, plasma E2, and P were performed at the anticipated time of ovulation based on at least two previous basal body temperature charts and menstrual patterns. RESULTS In untreated women there was a very strong tendency for CM quality to improve with rising plasma E2 levels and to worsen with rising plasma P levels. There was a significant association between CM quality and PCT results. Similar results were found in CC-treated women, except that plasma E2 was very significantly higher and there was a significant inverse relationship between plasma E2 and CM quality. CONCLUSION High plasma E2 in the periovulatory phase in CC-treated women is a marker for increased sensitivity to and continuing action of the antiestrogen. This impairs the quality of the CM.

FAILURE TO SUPPRESS ADRENAL FUNCTION IN CONGENITAL ADRENAL HYPERPLASIA (2 1 ‐HYDROXYLA SE DEFICIENCY) THREE CASE REPORTS

Three patients, aged 14, 16, and 32 years respectively, with congenital adrenal hyperplasia (21 ‐hydroxylase deficiency) are described. Excessive adrenal activityand ACTH secretion could not be suppressed with doses of corticosteroids sufficient to cause iatrogenic Cushings syndrome, even though part of the steroid dosage was administered in the late evening. The resistance to feed‐back suppression was of the same order as that seen in Cushings syndrome. Adrenalectomy was performed in the 16‐year‐old girl, and was followed by a menarche. Adrenalectomy was considered inadvisable in the other two patients.

Effect of Cell Density on Hormonal Secretion from Human Pituitary Adenomas in vitro

Cell density effects were investigated on tumorous hormonal secretion from 10 pituitary adenomas: 3 somatotrophinomas secreting GH and PRL; 7 gonadotrophinomas, 3 co-secreted both FSH and LH, all 7 secreted LH. Enzymatically dispersed tissue was plated out in 24-well plates at 5 ×105, 105, 5 ×104 and 104 cells/well in serum-free media. Media were collected weekly for 2 weeks. Results: In 3 of 3 somatotrophinomas, GH and PRL secretion was higher (p < 0.05) at both week 1 and 2 from 104 cells/well, but similar at other cell densities. In all 3 gonadotrophinomas, the FSH secretory rate was highest at 5 ×105 cells/well which fell as cell density decreased. Conversely, in 7 of 7 gonadotrophinomas the LH secretory rate was highest at 104 cells/well (p < 0.01) which fell as cell density increased. Conclusion: These data suggest that paracrine factors may modulate tumorous GH, PRL, FSH and LH secretion, and show that FSH and LH secretion vary inversely as cell density increases.

Human anterior pituitary adenoma cell attachment in vitro

Dear Editor: The purpose of this letter is to inform fellow scientists of a method for the attachment of enzymatically dispersed human anterior pituitary adenoma cells, which often adhere poorly to the surface of cell culture dishes preventing dynamic in vitro studies: this is especially recognized for prolactin secreting and nonfunctioning pituitary adenomas (3,10-12). Attempts to enhance pituitary adenoma attachment have included the use of an extracellular matrix derived from bovine corneal endothelium, which has been reported to be successful for prolactinomas (3) and Cushings adenomas (15). It has also been reported that rat pituitary endocrine cells express laminin and collagen IV, and that these cells grow as small clusters on fibroblast monolayers, which in turn are composed of collagen IV, heparin sulphate, and glycoprotein (13). Others have shown that the extracellular matrix components present among epithelial ceils forming Rathkes pouch consist of laminin, fibronectin, and collagen IV (8). Therefore, we set out to determine the optimal conditions for the attachment of different human anterior pituitary adenoma subtypes in dispersed culture using a panel of attachment factors: an extracellular matrix derived from bovine corneal endothelial cells, collagen type I and IV, fibronectin, laminin, human extracellular matrix (HEM), poly-l-lysine, plastic, and glass. Sixteen human pituitary adenomas--three macroprolactinomas, eight acromegalic adenomas, four nonfunctional tumors, and one corticotrophinoma removed by transsphenoidal surgery--were dispersed to single cells using a combination of a 200 mOsm hypoosmolar medium, clostripain inhibited crude collagenase, and dispase type 2 (1). Adenoma cells were plated at a density of 10 ~ cells per well in plastic 24-well plates with and without attachment factors in Iscoves modified Dulbeccos medium (IMDM) supplemented with 10% fetal calf serum (FCS) and gentamicin (50 rag/l). Cultures were maintained at 37 ° C in a humidity controlled atmosphere of 95% air and 5% CO2. After the cells had been allowed to adhere for 4 d, the total attachment was assessed using a calibrated graticule and viability was determined using fluorescein and propidium iodide (2), following which the wells were gently washed three times in IMDM to remove nonattached cells, and attachment and viability were reassessed. Four replicates were used for each group. Bovine corneal endothelial matrix (BCEM) were prepared from cells 7 d postconfluence: 1-6 d postconfluence gave a BCEM that was too thin, contained multiple holes, and peeled off from the culture surface; 9 d postconfluence the membrane was thicker, but peeled off after preparation. Three methods were compared for BCEM preparation: firstly, BCE cells were washed with phosphate-buffered saline (PBS) and 5 ml of 0.5% Triton X100 (Sigma Chemical Co., St. Louis, MO) (6); secondly, 0.1% Triton X100 ]adapted from (6)]; or thirdly, 20 mmol freshly prepared ammonium hydroxide (5) was added and incubated for 15-20 min and monitored by phase contrast microscopy. When the nuclei and extracellular matrix became visihie, the flasks were washed three times with PBS and once with IMDM. Cells from each tumor were seeded onto plastic plates treated with BCEM, collagen type I and type I¥, fibronectin, laminin, human extracellular matrix, poly-l-lysine, 24 plates alone and new glass coverslips (detailed in Table 1). The resultant attachment (percent) to each surface/attachment factor and cell viability (percent) is shown in Table 1. These studies suggest that the BCEM may be a universal attachment method for human pituitary adenoma cells in vitro, although the technique is limited by its technical complexity and the fragility of the BCEM. All pituitary adenoma subtypes attached and were viable on the BCEM (range 20-96% attachment; viability range 5097%) and the results are in accord with BCEM attachment of prolactinomas and Cushings adenomas reported previously (2,15). The BCEM was superior to collagen type I and IV, fibronectin, laminin, HEM, poly-l-lysine, plastic, and glass. There was no difference between the BCEM preparation methods for the Triton X100 or the sodium hydroxide method (data not shown). The BCEM is composed mainly of type III and type IV collagen (14), but it has been suggested that other factors may also be present (6). The BCEM may also play an active role in pituitary function; prolactin secretion from rat GH3 ceils has been found to be higher on BCEM compared to plastic alone, and cells cultured on plastic secreted more prolactin in response to TRH stimulation than those cultured on BCEM (4,9). Although poly-l-lysine showed good adenoma cell attachment generally, cell viability was markedly diminished compared to the BCEM, suggesting that poly-l-lysine is not suitable for dispersed human anterior pituitary adenoma cell culture. Collagen IV appeared to be superior to collagen I for the attachment of adenoma cells, which may reflect the reports of rat endocrine epithelial cells and Rathkes pouch expressing collagen IV rather than collagen I (8,13). However, neither attached the 3 prolactinomas studied and collagen I attached only 6 of 13 tumors, while collagen IV attached only 12 of 13 tumors; there was no difference in viability between collagen I and collagen IV for attached tumors. The poor attachment of adenoma cel~s to fibronectin (attaching 6 of 13 tumors) and laminin (attaching 5 of 13 tumors) was unexpected because both of these components have been found in the basement membrane in rat endocrine cells and Rathkes pouch (8,13), suggesting that a combination of extracellular components may be required for attachment of pituitary adenoma cells in vitro. No adhesion to the commercial HEM preparation occurred; however, this preparation is composed of a high percentage of laminin to which pituitary cells were seen to attach poorly. Plastic alone attached all adenomas apart from the three prolactinomas and one nonfunctional adenoma; there was no difference in attachment between the three plastic surfaces examined.