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BACKGROUND: Ethical constraints limit the ability to study peri-implantation phase human endometrium. In this study, the donor oocyte model was used to study candidate endometrial markers of uterine receptivity. METHODS: Archived, paraffin-embedded tissue obtained by endometrial biopsy during cycle days 21–23 of patients undergoing `mock' hormonal treatment cycles were evaluated by standard histological criteria and immunohistochemical staining for αvβ3 integrin and glycodelin. All of these patients (n = 101) had undergone a donor oocyte embryo transfer cycle utilizing the exact same hormonal protocol. RESULTS: Histological evaluation revealed 62 (61.3%) in-phase, 34 (33.7%) dyssynchronous, 2 (2.0%) immature and 3 (3.0%) advanced endometria. The clinical outcomes of patients with either in-phase or dyssynchronous endometria were similar. Very strong correlations were noted between endometrial glandular dating and either αvβ3 integrin or glycodelin immunostaining intensity (P < 0.001 for both). Glycodelin and αvβ3 integrin immunostaining intensities were also highly correlated with each other (P < 0.001). CONCLUSIONS: Throughout the time period corresponding to the putative window of maximal endometrial receptivity (cycle days 21–23) a dynamic process was observed in exogenous hormonal replacement cycles characterized by a rapid histological advancement of endometrial glandular elements as well as progressive αvβ3 integrin and glycodelin expression.

Insulin-like growth factors IGF-I and IGF-II are bound to specific binding proteins in serum. The lower mol. wt binding protein (IGF-BP) has been detected in various tissues, including secretory endometrium and preovulatory follicles of the ovary. This group studied the circulating levels of IGF-BP in the serum of 23 patients with polycystic ovarian disease (PCOD) and found that one-third of them have a subnormal level. In comparison with PCOD patients with a normal level, those with a subnormal level had a higher degree of obesity and a tendency to be more hirsute. They also had a higher serum insulin concentration and testosterone/sex hormone-binding globulin (SHBG) ratio, but lower serum SHBG concentration than those with a normal IGF-BP level. PCOD is the second abnormal clinical condition, after insulinoma, in which subnormal serum IGF-BP concentrations have been reported. The significance of low serum IGF-BP levels to pathophysiology of PCOD remains to be elucidated by studies on local interaction between IGF-BP and insulin in the polycystic ovary.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

The mechanisms underlying the protection of the human embryo/fetus from the maternal immune response are poorly understood. Substantial evidence indicates that carbohydrate recognition plays a primary role in the sequestration of leukocytes during inflammatory processes, lymphocyte homing, and initial gamete binding. Our previous studies suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Our more recent findings indicate that oligosaccharides participating in such processes are also associated with soluble glycoconjugates found in the human placenta, amniotic fluid, and decidua. We theorize that such glycoconjugates may abrogate the maternal immune/inflammatory response by blocking the primary adhesive interactions required for the expression of such activities. Foreign embryonic cells may also be protected by surface expression of oligosaccharide sequences that suppress immune effector cell action in a manner not dependent upon classical major histocompatibility (MHC) recognition. Glycoconjugates expressing selectin ligands may also manifest a potent contraceptive effect that may also be beneficial for both the mother and the developing embryo/fetus. This hypothesis provides a preliminary framework for understanding how temporally and spatially restricted immunosuppressive effects could be expressed in utero that protect the human embryo/fetus during this period of human development.

Radioimmunoassay, gel filtration, isoelectric focusing, and immunoperoxidase methods were used to study the levels, properties, and localization of pregnancy-associated plasma protein A (PAPP-A) in the human seminal plasma and male genital tract. Seminal plasma specimens from 20 normospermic and 20 oligospermic men were studied. PAPP-A was found in 30 of 40 samples, the levels ranging from undetectable to 135 μg/liter (median 35 μg/liter) in the normospermic group and from undetectable to 111 μg/liter (median 21 μg/liter) in the oligospermic group. There was no significant difference in the PAPP-A levels between the two groups, and no correlation was observed between the seminal plasma PAPP-A levels and the volume of seminal plasma or the sperm count or the viability of spermatozoa. Serial dilutions of seminal plasma and PAPP-A standard yielded parallel dose-response curves, and incubation with seminal plasma did not result in any change in the elution pattern of [125I] PAPP-A in gel filtration. PAPP-A-immunoreactive material from seminal plasma eluted as two peaks, the major one corresponding to the elution volume of purified PAPP-A and the minor eluting more slowly. The isoelectric point of seminal plasma PAPP-A was 4.3-4.7 and that of term pregnancy serum was 4.2-4.6. In the immunoperoxidase staining, PAPP-A was seen in the epithelium of the prostate, seminal vesicle, and the ampullar part of the vas deferens but not in the testis, epididymis, proximal parts of vas deferens or urethra. The results confirm the occurrence of PAPP-A in the seminal plasma but do not suggest any clinical utility for seminal plasma PAPP-A measurements. Distal glandular parts of the genital tract are the site of PAPP-A localization in tissue, indicating either synthesis or uptake.

Placental protein 14 (PP14), also known as progestagen-dependent endometrial protein and pregnancy-associated endometrial alpha 2-globulin, is synthesized by the human secretory endometrium and decidua. We have isolated from a human decidual cDNA library clones corresponding to PP14 and deduced its entire amino acid sequence. PP14 contains 180 amino acids, 18 of which correspond to a putative signal peptide. The predicted molecular weight of the pre-PP14 is 20,555 and that of the mature protein is 18,787. PP14 is encoded by a 1-kilobase-pair mRNA that is expressed in human secretory endometrium and decidua but not in postmenopausal endometrium, placenta, liver, kidney, and adrenals. The 162-residue-long sequence of PP14 is highly homologous to beta-lactoglobulins, with a 53.4% identity with the amino acid sequence of horse beta-lactoglobulin I. The four cysteinyl residues (positions 66, 106, 119, and 160) responsible for intramolecular disulfide bridges in beta-lactoglobulins are all conserved in PP14. Southern blot analysis of human DNA suggested that PP14 gene sequences encompass some 20 kilobase pairs of the human genomic DNA.

Three of 24 infertile women with a short luteal phase transiently showed chorionic gonadotrophin (HCG) like immunoreactivity in serum as measured by the HCG-beta subunit radioimmunoassay. The plasma progesterone concentration was elevated above the lower normal post-ovulatory level in 2 of these women who also had elevated basal body temperature at the time HCG was detected. Positive HCG reaction coincided with the pre-ovulatory surge of luteinizing hormone (LH) in one patient, but in 2 other cases the LH concentration was low suggesting absence of LH interference. Our results suggest that some infertile women may conceive during their cycles with a short luteal phase and maintain pregnancy until HCG becomes detectable.

The effect of pyridoxine (B6) on galactorrhoea, amenorrhoea, and the galactorrhoea-amenorrhoea syndrome was studied in 22 patients. No consistent change in serum prolactin concentration and no resumption of ovulation were seen during 3 months B6therapy. Inappropriate lactation ceased in 3 of 10 patients with galactorrhoea alone, and menses were restored in 2 of 9 patients with the galactorrhoea-amenorrhoea syndrome while on B6therapy. Our results suggest that B6is not generally effective in the treatment of galactorrhoea-amenorrhoea syndromes.

Hyperprolactinaemia occurred in 40 of 123 unselected patients (33%) with functional secondary amenorrhoea. Galactorrhoea was present only in 15 of these patients (38 %). On the other hand, galactorrhoea was found in 10 of 83 euprolactinaemic patients (12%). The frequency and severity of hyperprolactinaemia and galactorrhoea dissociated in various forms of amenorrhoea. Anorexia nervosa was characterized by euprolactinaemia and the absence of galactorrhoea. Self-induced weight loss resulted in amenorrhoea with a moderate degree of hyperprolactinaemia in 23 % of the cases. Yet, galactorrhoea was uncommon (1.6 %). Amenorrhoea following stress was also associated with moderate hyperprolactinaemia in 31 % of patients and a low frequency of galactorrhoea. By contrast, both hyperprolactinaemia and galactorrhoea occurred more frequently (36 %) and were more severe in patients who had post-pill amenorrhoea, but still 50 % of hyperprolactinaemic patients had no galactorrhoea and 28 % of euprolactinaemic patients had galactorrhoea. This discordance was accentuated in those patients who had been pregnant even if pregnancy did not immediately precede amenorrhoea. Galactorrhoea (63 %) and hyperprolactinaemia (36 %) were common in these patients and very high serum prolactin values were sometimes seen. Our results point to different mechanisms involved in the regulation of hypothalamic pituitary function and breast secretion in various forms of secondary amenorrhoea, and they may help select patients in whom the prolactin assay is likely to identify indication for specific treatment.

Specific high-affinity low-capacity binding of human chorionic gonadotropin (hCG) and lutropin (hLH) was demonstrated in the plasma membrane fractions of human corpora lutea. The number of binding sites for both hormones increased from early to late luteal phase, whereas regressing corpus luteum from proliferative phase did not bind either hormone. On the basis of apparent dissociation constants the affinity of the receptor for hLH is highest during early luteal phase and decreases toward the end of the cycle, which may reflect an increasing insensitivity of the corpus luteum to circulating hLH. By contrast, the affinity of the receptor for hCG is highest in the midluteal phase. There are gonadotropin binding sites in the human corpus luteum also during pregnancy, but they are saturated by endogenous hCG. Evidence for this was obtained by elution of hCG with 0.15M sodium chloride at pH 2.3 from washed plasma membrane fractions of luteal tissue from six to 16 week's gestation. After acid treatment and neutralization these preparations showed specific binding for 125I-labeled hCG, but not for 125I-labeled hLH. Our results demonstrate a shift in the balance of affinity of the gonadotropin receptor from hLH to hCG during the course of luteal phase, and during pregnancy the binding sites appear to be available for hCG only.

Amniotic-fluid alpha-fetoprotein (A.F.P.) levels were raised in early pregnancy in association with anencephaly and "open" spina bifida. Closed lesions, including encephalocele and hydrocephalus, were associated with normal levels as was an "open" spina bifida at 33 weeks of gestation. Radioimmunoassay of maternal serum-A.F.P. gave results in the normal range in eight of nine cases of neural-tube malformations tested, including anencephalics and open spina bifidas. In only one case, in which an anencephalic fetus was spontaneously aborted, was the serum level unequivocally raised. It is concluded that when ultrasound and amniocentesis are used most fetuses with anencephaly and open spina bifida are detectable before 20 weeks of gestation allowing selective abortion of most cases with neurological involvement when there is a history of previous affected fetuses. Closed lesions will usually be missed, and maternal serum-A.F.P. assay cannot be relied upon to detect neural-tube malformations in early pregnancy, whether open or closed.

PSBG and hCG were estimated by radioimmunoassays in the serum of 19 consecutive patients undergoing abdominal surgery for suspected ectopic pregnancy. Ectopic pregnancy was verified at operation in 14 cases, and the other five patients had a hemorrhagic cyst of the corpus luteum. Demonstrable PSBG and hCG were found in all patients with ectopic pregnancy. One of them had taken bromocriptine for infertility, and in serial blood samples taken during the luteal phase both PSBG and hCG were identified on day 24 of the fertile cycle. One of the five patients with a hemorrhagic cyst of the corpus luteum showed detectable PSBG and hCG in several serum samples taken before operation. This patient had experienced legal abortion 3 months ago, and the presence of PSBG and hCG in her serum may represent prolonged trophoblastic activity after pregnancy. Our results show that PSBG appears in the serum very early in pregnancy. However, some obscure positive results may be encountered, and this must be appreciated when these markers are applied for clinical use. Since the PSBG and hCG tests are likely to have different sources of error, PSBG estimation may become a useful adjunct to the hCG test in the detection of trophoblastic activity in suspected ectopic pregnancy.

Intrauterine devices (IUDs) exert contraceptive action by interfering with sperm transport, ovum development, fertilization and implantation. Glycodelin A (GdA) is a uterine glycoprotein that has local contraceptive activity by inhibiting sperm-egg binding. GdA is normally absent from endometrium during the fertile midcycle and it is not expressed until the fifth postovulatory day. The phase of menstrual cycle addressed in this study covers the phase when conception is most likely to follow an unprotected intercourse and when GdA is normally absent. We present here evidence that levonorgestrel-releasing IUD (LNg-IUD) is accompanied by 'inappropriate' expression of GdA in endometrium between days 7 and 16 of the menstrual cycle (six out of six cases). The same was also found in copper-releasing IUD (Cu-IUD)-wearing women, but less frequently (four out of 11 cases, P < 0.0345, Fisher's exact test). In-situ hybridization localized GdA mRNA into endometrial glands in the midcycle endometrium, confirming the cellular site of synthesis. Based on the potent inhibitory activity of GdA on sperm-egg binding, the presence of GdA in uterine glands of IUD wearers may lead to prior exposure of sperm to contraceptive GdA, thus contributing to the contraceptive activity of the IUD.

Immunofluorescence staining was used to localize two 'endometrial' proteins, insulin-like growth factor-binding protein and human beta-lactoglobulin homologue, in cultured cells of chorionic villi and decidua. Both cultured cell types were positive for each protein. These results indicate that immunohistochemical detection of these two proteins cannot be used to distinguish between cultured chorionic villus and decidual cells.

Luteinizing hormone (LH) responses to luteinizing hormone releasing hormone (LHRH), and growth hormone (GH) and cortisol responses to insulin induced hypoglycaemia were studied in 56 women classified into 4 distinct groups of functional secondary amenorrhoea. The groups were: I, self-induced weight reduction (20 patients); II, post pill amenorrhoea (14 patients); III, anorexia nervosa (10 patients); and IV, idiopathic secondary amenorrhoea (12 patients). Only patients with no overlapping anamnestic factors were included.Group Ipatients had the most heavily impaired LHRH-LH responses, and the GH response to hypoglycaemia was smaller than in other groups. Cortisol responses were normal. Group IIpatients showed blunted LH responses and normal GH and cortisol responses. Group IIIpatients showed normal or exaggerated LH responses in the recovery phase of anorexia nervosa, while those two patients who were in the static phase of the illness had impaired responses. GH responses varied greatly. Group IVpatients had normal basal levels of LH and normal LH, GH and cortisol responses.The restoration of LH response is not solely correlated to body mass, since patients recovering from anorexia nervosa showed greater LHRH-LH responses with nutritional rehabilitation at 76% of ideal body weight than patients with self-induced weight reduction at 87% of ideal body weight. In idiopathic amenorrhoea the hypothalamic pituitary axis seems to be practically intact.The function of hypothalamic-pituitary axis may be impaired selectively in functional amenorrhoea. Corticotrophin releasing hormone function remains intact, and GH-response may be impaired or normal independently of the LH-response to LHRH. In self-induced weight reduction both functions were impaired. These tests are easily carried out with outpatients, and they give more information about the functional state of hypothalamic-pituitary axis than basal analyses of gonadotrophins and oestrogens. However, a single pathologic reading in the LH response is not specific enough to indicate to which group of amenorrhoea the patients belong, but these tests together elucidate the severity of lesion in hypothalamic pituitary axis.

The effects of low daily doses of the antiprogestin mifepristone (RU 486) on ovarian and endometrial function were studied. The study included one control cycle, three treatment cycles and one follow-up cycle. During the treatment cycles, either 0.1 (n = 5) or 0.5 (n = 5) mg of mifepristone was administered once daily. Urine samples were collected three times weekly during the control and treatment cycles and pregnanediol glucuronide and oestrone glucuronide and luteinizing hormone (LH) were quantified by radioimmunoassay. Blood samples for cortisol measurement were collected once weekly and for serum glycodelin at the onset of menstruation. An endometrial biopsy was obtained in the mid-luteal phase in the control cycle and in the first and third treatment cycles and analysed by morphometric and histochemical methods. Binding of Dolichus biflorus agglutinin (DBA) lectin was measured and expression of progesterone and oestrogen receptors and glycodelin were analysed immunohistochemically. All cycles studied were ovulatory with an LH peak and elevated pregnanediol glucuronide concentrations. Follicular development seemed normal as judged by ultrasound examination. The length of the menstrual cycle and the menstrual bleeding were not significantly altered. Following administration of 0.5 mg mifepristone/day, endometrial development appeared to be slightly retarded and glandular diameter was significantly reduced. Furthermore, significant decreases in DBA lectin binding and endometrial expression of glycodelin were observed. Daily doses of 0.1 mg did not have any significant effect on the endometrium. No differences in oestrogen or progesterone receptor immunoactivity between control and treatment cycles were seen. This study provides further evidence that endometrial function is sensitive even to doses of antiprogestin that are low enough not to disturb ovulation. It remains to be established whether these effects are sufficient to prevent implantation.

Alpha fetoprotein (AFP) was determined by radioimmunoassay in 65 amniotic fluid samples. During the second trimester, the AFP level in amniotic fluid varied from 2,800 to 26,000 nanograms per milliliter; and, during the third trimester, from 15 to 535 ng. per milliliter. A correlation between advancing gestation and the decreasing AFP level during the third trimester was found to be statistically highly significant (r = −0.695; p < 0.001). In a case where the infant had a congenital nephrosis, the AFP level in amniotic fluid was 4,900 ng. per milliliter at 36 weeks. Fetal urine during the second trimester contained 5,900 to 48,000 ng. of AFP, and the level in newborn urine at birth was 2.5 to 21 ng. per milliliter. These data suggest that the assay of amniotic fluid AFP might prove useful in estimation of gestational age of the fetus.

The alpha fetoprotein (α-FP) concentration in uterine hemorrhage was studied by radioimmunoassay in 71 samples from 69 women at different stages of pregnancy and delivery. Thirty-eight women had a diagnosis of imminent abortion: In 34 women who aborted, the medium α-FP concentration in uterine bleeding was 1,675 ng. per milliliter (mean 2,263 ng. per milliliter), whereas in 4 women whose pregnancy continued to term the median α-FP level was 407 ng. per milliliter. In 6 women with premature separation of the placenta, the α-FP concentration in bleeding was higher (median 805 ng. per milliliter) than in 5 women with placenta previa (median 215 ng. per milliliter) or 20 women with a nonclassified late pregnancy hemorrhage (median 235 ng. per milliliter). The result indicates that bleeding from the fetus occurs to a greater extent in premature separation of the placenta than in the other late pregnancy hemorrhages studied. The clinical use of the α-FP test in cases of imminent abortion and antenatal hemorrhage could be valuable in predicting the prognosis of the pregnancy and the infant.