The effects of sex steroid hormones and interleukin-1-beta on MUC1 expression in endometrial epithelial cell lines.

Oestrogen, progesterone and paracrine signals from the embryo have been associated with the overall control of implantation. Changes in the expression of the heavily glycosylated transmembrane glycoprotein MUC1 mucin on the endometrial epithelium are also thought to be important for embryo attachment. Increased MUC1 expression has been correlated with elevated progesterone levels in the secretory phase of the menstrual cycle. Embryonic control of endometrial receptivity through changes in MUC1 expression could be achieved through the interleukin-1 system. Four endometrial epithelial cell lines (HEC1A, HEC1B, Ishikawa and RL592) were treated with oestrogen and progesterone (with or without interleukin-1-beta) and were subjected to immunocytochemistry and flow cytometric analysis to determine MUC1 production using MUC1 antibodies. HEC1A (oestrogen receptor (ER) and progesterone receptor (PR) positive) and HEC1B (ER positive and PR negative) were transfected with the MUC1 promoter, underwent similar treatment regimes and the activity of the MUC1 promoter relative to their untreated controls was determined using a chloramphenicol acetyltransferase (CAT) enzyme-linked immunoassay. Using the cell lines, we determined that endometrial MUC1 expression is up-regulated by progesterone, consistent with the in vivo increases in MUC1 related to high progesterone levels. We also revealed that neither oestrogen, nor interleukin-1-beta, appear to modulate MUC1. Progesterone-dependent regulation of MUC1 is likely to be an important factor in determining endometrial receptivity.

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women.

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

MUC 1: a genetic susceptibility to infertility?

In man and some animals regulation of embryo implantation by endometrial expression of the highly polymorphic MUC 1 mucin has been suggested. We assessed the polymorphism of MUC 1 in women known to be fertile and those with infertility due to suspected failure of embryo implantation. The median of the lower allele size in the infertile group was only 2.5 kb compared with 3.4 kb in the fertile group (p=0.0029, difference 0.9, [95% CI 0.1-1.3]). Women with unexplained infertility might have a genetic susceptibility to failure of embryo implantation due to small MUC 1 allele size.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE: To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN: Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING: Patients giving informed consent in hospitals. PATIENT(S): Women undergoing laparotomy or oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular size, E2, histology. RESULT(S): Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S): It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells.

OBJECTIVE: Locally produced oestrogens and prostaglandins (PGs) are implicated in the regulation of luteal lifespan in the human ovary. This study (1) assesses direct effects of these factors on progesterone synthesis in isolated luteal cells, and (2) explores interactions between luteal age and treatment with gonadotrophin or oestrogen on the metabolism of arachidonic acid (prostaglandin precursor) by steroidogenic luteal cells in vitro. DESIGN: Primary monolayer cultures of human luteal cells obtained at different stages of the luteal phase were used to investigate the effect of oestradiol, catechol oestrogens (2- and 4-hydroxyoestradiol), diethylstilboestrol, PGE2 and PGF2 alpha on basal and human chorionic gonadotrophin (hCG) stimulated progesterone production in vitro. The role of PGs as modulators of luteal cell function was further investigated by studying the metabolic fate of radioactively labelled arachidonic acid in hormone treated (oestradiol and hCG) and control cultures, assessed by high performance liquid chromatography. PATIENTS: Corpora lutea were enucleated from nine women with regular ovulatory cycles undergoing microsurgical reversal of tubal sterilization. Granulosa cell aspirates were obtained from three patients undergoing in-vitro fertilization treatment. RESULTS: PGE2 and PGF2 alpha at various concentrations did not have a consistent effect, whereas oestradiol, diethylstilboestrol (and 2-hydroxyoestradiol in early luteal cell cultures) significantly inhibited basal and hCG stimulated progesterone biosynthesis. Evidence for direct inhibition of 3 beta-hydroxysteroid dehydrogenase enzymic activity by oestradiol was obtained. Both major metabolic pathways of arachidonic acid (lipoxygenase and cyclo-oxygenase) were operative in steroidogenic luteal cells recovered throughout the luteal phase. The ratio of PGE2 to PGF2 alpha synthesis in vitro by human luteal cells from endogenously incorporated arachidonic acid did not change significantly with corpus luteum age, with PGE2 tending to predominate. Oestradiol treatment shifted arachidonic acid metabolism from the lipoxygenase towards the cyclooxygenase pathway in cells isolated from ageing corpora lutea. CONCLUSIONS: Oestradiol, at relatively high concentrations, is a potent inhibitor of basal and hCG induced luteal cell steroidogenesis in vitro. No support is provided for the concept that luteolysis is mediated by local production of PGF2 alpha. The putative luteolytic effect of oestradiol may entail reduced metabolism of arachidonic acid to lipoxygenase derived products by luteal cells rather than direct stimulation of prostaglandin production by itself.

Microsurgical salpingostomy is not an obsolete procedure.

OBJECTIVE: A review of the results of microsurgery for bilateral distal tubal blockage. DESIGN: A retrospective review. SETTING: Hammersmith Hospital London and local private hospitals. SUBJECTS: 388 patients with bilateral ampullary occlusion treated between 1971 and 1988 by microsurgery. INTERVENTIONS: Full investigation for other causes of infertility followed by abdominal microsurgical salpingostomy. Repeated meticulous follow-up was essential with check laparoscopy one year after surgery. MAIN OUTCOME MEASURE: Successful pregnancy in relation to tubal damage. RESULTS: In 65 women microsurgery followed tubal reocclusion after failed conventionally performed salpingostomy. 74 women (23%) had one term pregnancy after primary salpingostomy and 12 women (18%) after repeat salpingostomy. Over half the women having a term pregnancy subsequently had a second infant. The tubal damage was classified in four stages according to the degree of mucosal damage and tubal fibrosis, the presence of isthmic disease and the quality of tubal and ovarian adhesions. Approximately one quarter of patients had stage I disease and amongst these 39% had babies after primary salpingostomy and 25% after repeat salpingostomy. CONCLUSION: Microsurgical salpingostomy is a specialized procedure. Proper selection of patients, competent microsurgical technique and adequate follow-up appear crucial to success. In selected patients treatment by salpingostomy gives better results than multiple cycles of in vitro fertilization.

Cellular basis of luteal steroidogenesis in the human ovary.

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20 alpha-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.

Improvement of in vitro fertilisation after treatment with buserelin, an agonist of luteinising hormone releasing hormone.

Treatment with buserelin, an agonist of luteinising hormone releasing hormone, and human menopausal gonadotrophin was compared with the conventional treatment of clomiphene citrate and human menopausal gonadotrophin in the outcome of in vitro fertilisation. Seventy seven infertile women had 83 cycles of treatment with buserelin and human menopausal gonadotrophin, and concurrently another 328 infertile women were treated with clomiphene citrate and human menopausal gonadotrophin. Seven (8%) cycles were cancelled owing to inadequate super-ovulation or ovarian hyperstimulation in the women receiving buserelin and 103 (31%) were cancelled because of poor follicular development in those receiving clomiphene citrate. The mean number of oocytes recovered was significantly higher with buserelin (9.5 (SD 4.5) v 5.5 (2.2)) as was the mean number of embryos obtained (4.3 (2.4) v 2.9 (1.7)). Significantly more women who had an embryo transfer became clinically pregnant after treatment with buserelin (53% (30/57) v 30% (48/159), or 36% v 14% of treatment cycles). Altogether 33% (10) of pregnancies in women treated with buserelin were multiple compared with 23% (11) in those treated conventionally. Of the 17 completed pregnancies in women treated with buserelin, 11 resulted in the birth of live babies (eight singletons, two sets of twins, and one set of triplets) and six failed, five before 12 weeks' gestation and one at 22 weeks. The 13 continuing pregnancies (32 weeks) were eight singletons, two sets of twins, and three sets of triplets. Of the 48 completed pregnancies in women treated with clomiphene citrate, 35 resulted in the birth of live babies (26 singletons, five sets of twins and four sets of triplets) and 13 failed, eleven before 12 weeks' gestation and two by 27 weeks. Buserelin increased the chance of pregnancy after in vitro fertilisation compared with conventional treatment, but the risk of multiple pregnancy may be increased.

Gonadotrophic control of human granulosa cell glycolysis.

Follicular fluid lactate levels were measured in women undergoing infertility surgery during the follicular phase or oocyte recovery for in-vitro fertilization (IVF). In the largest ovulatory follicle lactate levels were low in the mid-follicular phase (group 1), 1.6-fold higher just prior to the onset of the luteinizing hormone (LH) surge (group 2) and a further 2.5-fold higher after the onset of the LH surge (group 3). In IVF patients mean lactate levels in all aspirated follicles were similar to those in group 3 subjects, but the levels within each patient were variable and were positively correlated with follicular volume. Basal granulosa cell lactate accumulation in vitro was 3-fold higher in group 3 compared with group 2 subjects, but stimulation by FSH or HCG was higher in group 2 (2- to 3-fold) compared with group 3 (1.4- to 2-fold). These results demonstrate that human follicular fluid lactate levels increase as a function of the maturity and size of the developing follicle. Granulosa cell lactate accumulation in vitro is under gonadotrophic control, which suggests that the effects observed in vivo reflect changes in granulosa cell glycolysis in response to gonadotrophic stimulation. Our findings support the concept that low molecular weight energy metabolites transduce gonadotrophin signals that regulate oocyte maturation.

Primary and secondary closure of ampullary salpingotomy compared in the rabbit.

Because there is disagreement as to how linear ampullary salpingotomy should heal, primarily or secondarily, we compared the two methods with the use of rabbit oviducts in which conditions simulating ectopic pregnancy had been induced. Pregnancy rates (86% versus 75%), nidation indices (62% versus 54%), and percentage of adhesion-free tubes (50% versus 32%) were greater after primary closure, but these differences were not statistically significant. Ampullary tuboperitoneal fistulae occurred at the site of operation in 6.8% of tubes and impaired fertility (P less than 0.005).

Superovulation strategy before in vitro fertilization.

This chapter reviews current understanding of the control of spontaneous preovulatory follicular development in the natural ovarian cycle as a basis for the design and use of superovulation strategy before clinical IVF. The principles, limitations and practical aims of therapy using clomiphene citrate and HMG to stimulate multiple follicular development are outlined together with details of methods in current use to monitor ovarian response to these drugs and to time ovulation induction and egg collection with HCG. Examples of successful IVF treatment cycles are given. It is stressed that properly controlled clinical trials to judge the relative merits of the various superovulation methods in current use for IVF have not been undertaken. Possible new approaches to ovarian stimulation before IVF include the use of 'pure' FSH, LHRH and pulsatile gonadotrophin administration.

Transperitoneal migration and ovum capture in rabbits.

It is not fully understood how eggs are captured by the fallopian tube. The close proximity of tube and ovary, and the movements of the fimbria across the ovarian surface during ovulation, are believed to contribute to ovum pickup. We studied the efficiency of ovum capture when contact between fimbria and ovary is prevented and the extent to which ovum transmigration may contribute to fertility. Unilateral right oophorectomy was performed in 55 rabbits, which were divided into three groups: in Group A, the contralateral oviduct was left intact; in Group B, the ampulla of the contralateral oviduct was occluded; in Group C, contralateral total salpingectomy was performed. None of the rabbits in Group A conceived on the oophorectomized side. Seven of 19 animals became pregnant in Group B and 15 of 18 conceived in Group C. The results indicate that direct fimbrial-ovarian contact is not essential for ovum capture, and that ovum transmigration could contribute to fertility.

Technique and results of ovarian microsurgery in preparation for in vitro fertilization.

A microsurgical technique was used to free the ovaries in women with severe adnexal adhesions, specifically to facilitate subsequent egg retrieval for in vitro fertilization. The surgical technique is described in detail. Eighteen of these patients had a single attempt at laparoscopic collection of oocytes. From 15 women in whom ovariolysis had been successful, 27 eggs were obtained (mean 1.8 eggs per patient). Three conceptions occurred following embryo transfer and one other patient conceived spontaneously. These results suggest that ovariolysis before extra-corporeal fertilization is worthwhile in selected cases.

Granulosa cell steroidogenesis before in vitro fertilization.

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with "over-ripeness" and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.

Serum oestradiol and preovulatory follicular development before in-vitro fertilization.

Twenty-three in-vitro fertilization (IVF) treatment cycles (four unstimulated and 19 clomiphene-stimulated) were assessed retrospectively to discern relationships among serum oestradiol (OE2) titre on the day that human chorionic gonadotrophin (hCG) was given and the number and size of ovulatory follicles available for aspiration of oocytes during laparoscopy 32-38 h after hCG injection. Since 12 of the cycles succeeded to the stage of embryo replacement and two normal term pregnancies resulted, the series as a whole offers a useful referent data base. When only one ovulatory follicle developed (n = 8) the average volume of aspirated follicular fluid was approximately 6 ml, equivalent to a follicular diameter between 22 and 23 mm. When multiple follicles developed (mean 2.7/patient, n = 15), average fluid volume/follicle was not significantly different, averaging approximately 5.5 ml. Serum OE2 titre on the morning before hCG was injected ranged between 0.9 and 5.5 nmol/l and corresponded to the number of follicles aspirated at laparoscopy. There was a highly significant linear correlation (r = 0.85, P less than 0.001) between this OE2 value (X nmol/l) and total aspirated fluid volume (Y ml) where Y = 2.07 + 3.65 X. Thus taking 6 ml as the 'typical' fluid volume, the calibration line and its 95% confidence limits could be used to establish provisional 'ideal' pre-hCG serum OE2 titre ranges corresponding to the development of one, two or three mature ovulatory follicles. This information, combined with a knowledge of the number of presumptive preovulatory follicles present (assessed by ovarian ultrasound), can aid the timing of the hCG injection before IVF.