Gerson Weiss

Myometrial inhibiting activity of relaxin-containing extracts of human corpora lutea of pregnancy

Relaxin is a peptide hormone secreted by the human corpus luteum of pregnancy . Aqueous extracts of relaxin-rich pregnancy corpora lutea decreased the amplitude of spontaneous human myometrial strip contractions in vitro. Relaxin-poor extracts of menstrual cycle corpora lutea did not affect contractions. Antibody precipitation of relaxin from pregnancy luteal extracts eliminated the effect on myometrial strips. Relaxin removal was confirmed by bioassay. This demonstrates an inhibiting action of human relaxin on human myometrial tissue in vitro. This action suggests a mechanism for maintaining uterine quiesence in early pregnancy.

Clomiphene citrate in the management of infertility associated with shortened luteal phases.

Repetitively short luteal phases were found in eight infertile women. The short luteal phase was defined as 10 days or less from the presumed time of ovulation (as assessed by basal body temperature recording) to the onset of menses. Clomiphene citrate (Clomid) therapy resulted in pregnancy in two patients and lengthened the luteal phase in the other six. Ultimately, seven of eight patients conceived during Clomid therapy. Clomid therapy can lengthen the luteal phase in patients with luteal temperature elevation of 10 days or less. The occurrence of short luteal phases may be associated with infertility.

Partial purification of relaxin from human seminal plasma

Human seminal plasma contains approximately 50 ng/ml of relaxin (specific activity = 1.3 ng/mg protein). During preliminary purification, semen plasma was delipidated, rehydrated, and loaded onto small octadecyl silica columns that were eluted with a TEAF/acetonitrile gradient system. Results were monitored by radioimmunoassay. The resultant partially purified human relaxin demonstrated biologic activity in the rat uterine segment bioassay. Nine liters of semen plasma was delipidated, rehydrated in TEAF, and subjected to high-performance liquid chromatography by a step gradient followed by a linear gradient. The active eluate was further purified by ion exchange chromatography. Pooled recovery fractions provided a total of 45.8 micrograms of relaxin. An aliquot flash evaporated and desalted by gel filtration chromatography provided 1.85 micrograms of relaxin in 25.2 mg protein, specific activity 73.4. This material is being used as immunogen in the production of antihuman relaxin antibodies by monoclonal technique. Our procedure represents the first and only successful partial purification of human relaxin to yield sufficient quantity and purity for antibody production.

SYNERGISTIC EFFECT OF HUMAN RELAXIN AND PROGESTERONE ON HUMAN MYOMETRIAL CONTRACTIONS

In an in vitro human myometrial strip system, both relaxin and progesterone can independently decrease the amplitude of spontaneous myometrial contractions. However, progesterone and relaxin synergize in this action. Doses of relaxin and progesterone which independently are ineffective, together inhibit myometrial contraction amplitude. Relaxin and progesterone are both products of the corpus luteum, a structure necessary for early pregnancy maintenance. The synergistic action of relaxin and progesterone in vitro suggests a similar in vivo physiologic effect in establishing uterine quiescence.

The effect of relaxin and progesterone on rat uterine contractions

The effect of porcine relaxin on electrically stimulated in vitro contractions of isolated uterine horn segments from estrogen-pretreated immature rats was studied. Relaxin decreased the amplitude of contractions. A mean of 8.3 ng/ml of relaxin produced a 90% decrease in contraction amplitude. There was a minimal effect of 1.0 microgram/ml of progesterone on contraction amplitude. In vitro pretreatment of the isolated uterine segment with this dose of progesterone for 15 minutes did not significantly affect the dose of relaxin needed to decrease the amplitude of contractions. In contrast, pretreatment with progesterone for 45 minutes significantly decreased the concentration of relaxin needed to decrease contraction amplitude. Only 4.7 ng/ml of relaxin was needed to produce a 90% decrease in amplitude after progesterone pretreatment for 45 minutes (p less than 0.005). Relaxin and progesterone synergize in decreasing the amplitude of uterine contractions in vitro. A similar effect may occur in vivo.

The Fibrinolytic Enzyme System in Cervical Mucus

n A clinical study was undertaken to assess the fibrinolytic enzyme system in cervical mucus in relation to the different stages of the menstrual cycle. Samples of cervical mucus from 8 women in the reproductive stage were taken on different days of the cycle and tested for components of the fibrinolytic system. The testing procedure is explained and the results graphed. Activator was found to disappear from the mucus for 2 days pre- and post-ovulation. Heating and freezing the mucus did not destroy the fibrinolytic activity. Testing was conducted for the presence of such other agents as proactivator, plasmin, and plasminogen in the mucus.n

Bioassay Methods for Relaxin: Uses and Pitfalls

The ability of aqueous extracts of porcine corpora lutea of pregnancy to “relax” the pubic symphysis of the estrogen-primed ovariectomized guinea pig was first described by Hisaw (1926). Subsequent work showed that porcine luteal extracts which contained guinea pig pubic symphysis-relaxing activity could inhibit contractions of uterine myometrium of rats in vitro (Sawyer et al. 1953) and guinea pigs in vivo (Krantz et al.3 1950). Around the same time, it was reported that crude extracts of sow corpora lutea induced interpubic ligament formation in estrogen primed mice (Hall and Newton, 1946; Kliman et al. 3 1953). These basic observations led to the development of numerous bioassay methods for relaxin which have been amply documented in previous reviews (Frieden and Hisaw, 1953; Hall, 1960; Steinetz et al. 1969). The present paper will, therefore, be devoted primarily to a discussion of the uses (and abuses!) of the various assay methods, the pitfalls which may be encountered in assaying relaxin activity in impure extracts and the growing realization that relaxin activity may be associated with a family of related polypeptides. Of special interest are recent observations which suggest that relaxin-like hormones obtained from different species may not be equally effective in the classical bioassay methods.

Localization of fibrinolytic activity in uterine cancer

Four specimens of invasive endometrial adenocarcinoma and five specimens of locally invasive squamous cell carcinoma of the cervix were studied for fibrinolytic activity by Todd’s histochemical technique. Only squamous cell cancer revealed activator activity. This was associated with the advancing front of the tumor and with local metastasis. Adenocarcinoma of the endometrium showed activity comparable to that of proliferate endometrium.

Progesterone and relaxin secretion in relation to the ultrastructure of human luteal cells in culture: effects of human chorionic gonadotropin

This report describes the first study to correlate the ultrastructure of long-term monolayer cultures of human luteal cells with their secretion of relaxin and progesterone under basal and human chorionic gonadotropin-stimulated conditions. In culture from 14 to 28 days, cells from both corpora lutea of the menstrual cycle and corpora lutea of pregnancy took on characteristics of granulosa luteal cells, particularly after exposure to human chorionic gonadotropin. Relaxin was detectable in the luteal cell cultures only at early time points. The effect of human chorionic gonadotropin on media relaxin levels differed in cells of the cycle and cells of pregnancy. In the cells of the cycle, relaxin was detectable only on day 2 and was decreased by human chorionic gonadotropin (p less than 0.01). In cells of pregnancy, relaxin was detectable for the first 4 days of culture and was not affected by human chorionic gonadotropin. Progesterone was detectable in all the luteal cell cultures and was enhanced by human chorionic gonadotropin (10 and 50 IU/ml) after 24 days of exposure. At earlier time points in both the luteal cells of the cycle and the luteal cells of pregnancy, the human chorionic gonadotropin-induced increases in progesterone levels were not as consistent. However, in all cases of progesterone enhancement, smooth-surfaced endoplasmic reticulum was increased in the human chorionic gonadotropin-treated cells compared with corresponding controls, consistent with more active steroid production. In addition, gap junctions, considered to be responsive to trophic hormones, were increased in the treated cells. In conclusion, this long-term monolayer culture of human luteal cells, as monitored by ultrastructural and hormonal changes, retained the differentiated function of progesterone secretion and exhibited responsiveness to human chorionic gonadotropin. Therefore, morphologic and functional aspects of progesterone secretion may be investigated more closely with use of this long-term luteal cell culture system.

Serum Unconjugated Estriol in the Menstrual Cycle and Early Pregnancy

A radioimmunoassay for serum unconjugated estriol in the menstrual cycle with a sensitivity of about 5 pg/ml is described. 8 cycles were studied. In 2 cycles, single spikes of 22 and 30 pg/ml were obtained. In 3 cycles, concentrations of 4-5 pg/ml were found whereas in the other 3 studies, no estriol was detected. In general, peaks of estriol corresponded to peaks in estradiol plus estrone. Patients in 4-12 weeks of gestation were also studied. Concentrations as high as 262 pg/ml were found but in isolated instances, no estriol was detected. The results support the view that in contrast to the pregnant state, in the normal menstrual cycle, the bulk of the estriol produced is conjugated before release into the blood.