Human Pituitary Gonadotropin Research Articles

O-279 The type of gonadotrophin: Does it improve ART outcome?

Abstract Ovarian stimulation is a vital clinical component of an IVF cycle, with gonadotrophins being the main stimulation drugs. Follicle stimulating hormone (FSH), the key driver of follicle recruitment during an ovarian cycle is a complex glycosylated protein comprised of an α and β subunit with four glycosylation sites. Differences in glycosylation lead to heterogeneity with the FSH isoforms varying in their receptor binding and biological activity. The cyclic physiological variation of FSH isoforms and the FSH threshold/ window concept lead to the selection of a single dominant follicle during a menstrual cycle. Surpassing the FSH threshold and extension of the FSH window with ovarian stimulation using gonadotrophins salvages follicles with higher FSH sensitivity leading to multi-follicular development and retrieval of multiple oocytes. The first IVF birth in 1978 was from a natural cycle and was preceded by several unsuccessful attempts at oocyte retrieval. Ovarian stimulation was therefore incorporated into IVF treatment to overcome the inefficiencies encountered with a natural cycle. The objective of ovarian stimulation during IVF treatment is to obtain several mature oocytes, leading to good quality embryos for immediate replacement and cryopreservation for future use. An ideal stimulation wherein gonadotrophins are the mainstay is one that achieves maximal efficacy, safety and cost-effectiveness. Several gonadotrophin preparations have been available for clinical use over the years since the first use of pregnant mare serum gonadotrophin (PMSG) in 1940 and the first successful birth using human pituitary gonadotrophin (hPG) in 1961. These initial preparations were soon abandoned due to concerns with antigenicity and safety. Subsequent important milestones in the development gonadotrophins for clinical use include the urinary derived products, recombinant FSH preparations, long acting FSH and the biosimilars. Given the selection of gonadotrophins currently available, the apparent question is if they differ? Circulating FSH isoforms change during different stages of the menstrual cycle and through the reproductive stages. More acidic isoforms are predominant during the early follicular phase, with less acidic or basic isoforms predominant in the late follicular phase of the menstrual cycle, and more acidic isoforms during the menopausal stage. Similarly, the different FSH products vary in their properties owing to differences in their production and purification. Urinary derived products are more acidic compared to recombinant preparations and the different recombinant products vary in degree of acidity due to differences in the extent and pattern of glycosylation. The acidic and basic properties of gonadotrophin types influence metabolic stability, half-life and their biological activity. The question then remains if these differences in bioactivity affect clinical outcomes? Clinical effectiveness of the various gonadotrophins types should be addressed in the context of the different pituitary suppression regimens, ovarian response categories and patient variables given the implementation of individualised stimulation protocols. It is also imperative to clearly define successful clinical outcomes incorporating both efficacy and safety. Cumulative live birth rate (CLBR) following fresh and frozen embryo transfers resulting from one episode of ovarian stimulation should be adopted as the measure of the overall efficiency along with OHSS as a measure of immediate safety. There are several studies and systematic reviews comparing the different gonadotrophin types and products for various outcomes including surrogate endpoints, with inconsistent results attributed to differences in the inclusion/ exclusion criteria, heterogeneity across studies and varied quality of the primary studies. Furthermore, there have been sparse studies reporting on CLBR and there is a continuing debate on what would be considered as a relevant clinical difference in efficacy. In addition to efficacy and safety, the ultimate choice of gonadotrophin should be based on regional availability, patient acceptability and cost-effectiveness for a live birth.

Analysis of somatic and reproductive history in patients with prognostically poor response of the testicles to gonadotropin stimulation

The objective: to conduct a clinical and statistical analysis of the somatic and reproductive history in women with infertility and a predictable poor ovarian response to stimulation with different gonadotropins. Materials and methods. We examined 140 infertile patients 21–42 years old with an increased risk of poor response. I group included 75 women who received gonadotropins of human pituitary origin in stimulated cycles, II group involved 65 patients who received recombinant gonadotropins in stimulated cycles. The analysis of the anamnestic data included an assessment of the menstrual cycle (age of menarche onset, parameters, duration, and regularity), characteristics of reproductive function (number of pregnancies, peculiarities of the course and outcomes of pregnancy), gynecological diseases, conservative and surgical treatment of infertility and its effectiveness. Somatic pathology in women was analyzed. Data processing was carried out using the Stat Soft Statistica 8.0 software package.Results. The average age of patients in the I group was 36.4±3.6 years, in the II group – 35.6±2.5 years. The normal menstrual cycle was in 89.3% of women in the I group and 92.3% – II. Menstrual cycle disorders of the type of oligomenorrhea were observed in 8 (10.7%) and 5 (7.7%) patients, respectively. Primary infertility was diagnosed in 21 (28.0%) persons in the I group and 18 (27.0%) – II group, secondary infertility – 54 (72.0%) and 47 (72.3%) women, respectively. The average duration of primary infertility was 6.2±2.1 years, secondary one – 5.8±3.8 years. The history of artificial abortion had 17 (22.7%) and 14 (21.5%) women, spontaneous abortion and missed pregnancy till 12 weeks of gestation were observed with the same frequency – 18 (24.0%) and 15 (22.0%) cases, respectively, ectopic tubal pregnancy – 9 (12.0%) and 7 (10.7%) cases. The rate of insufficient ovarian function was determined in 49 (65.3%) women in the I group and 42 (64.6%) – the II group; it was manifested by chronic anovulation in 89 women of both groups, which accounted for 63.5% of patients and insufficiency of the corpus luteum (38 (27.1%) of the examined persons). Pelvic inflammatory diseases were diagnosed in 41 (54.6%) and 36 (55.4%) patients, external genital endometriosis – 22 (29.3%) and 17 (26.1%) women, respectively. Ovarian resection was performed in 21 (28.0%) patients of the I group and 16 (24.6%) – II group, the indication for which in 57.1% and 56.3%, respectively, was a recurrent course of external genital endometriosis with the formation of endometrioid cyst. The pathological changes in the endometrium were detected in 39 (52.0%) and 31 (47.7%) patients of the I and II groups, respectively, among which 14 (18.6%) and 11 (16.9%) women had polyps, 11 (14.7%) and 9 (13.8%) – chronic endometritis. Conclusions. Thus, the predisposing factors for the development of a poor response in vitro fertilization programs were late reproductive age, a high frequency of surgical interventions on the pelvic organs and, as a result, a change in the blood circulation of the ovarian cortex, a decrease in the volume of the stroma and a reduction in the follicular reserve. According to the main clinical and anamnestic characteristics, there were no significant differences between patients who received human pituitary or recombinant gonadotropins in stimulated cycles.

The Development of Gonadotropins for Clinical Use in the Treatment of Infertility.

The first commercially available gonadotropin product was a human chorionic gonadotropin (hCG) extract, followed by animal pituitary gonadotropin extracts. These extracts were effective, leading to the introduction of the two-step protocol, which involved ovarian stimulation using animal gonadotropins followed by ovulation triggering using hCG. However, ovarian response to animal gonadotropins was maintained for only a short period of time due to immune recognition. This prompted the development of human pituitary gonadotropins; however, supply problems, the risk for Creutzfeld–Jakob disease, and the advent of recombinant technology eventually led to the withdrawal of human pituitary gonadotropin from the market. Urinary human menopausal gonadotropin (hMG) preparations were also produced, with subsequent improvements in purification techniques enabling development of products with standardized proportions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activity. In 1962 the first reported pregnancy following ovulation stimulation with hMG and ovulation induction with hCG was described, and this product was later established as part of the standard protocol for ART. Improvements in immunopurification techniques enabled the removal of LH from hMG preparations; however, unidentified urinary protein contaminants remained a problem. Subsequently, monoclonal FSH antibodies were used to produce a highly purified FSH preparation containing <0.1 IU of LH activity and <5% unidentified urinary proteins, enabling the formulation of smaller injection volumes that could be administered subcutaneously rather than intramuscularly. Ongoing issues with gonadotropins derived from urine donations, including batch-to-batch variability and a finite donor supply, were overcome by the development of recombinant gonadotropin products. The first recombinant human FSH molecules received marketing approvals in 1995 (follitropin alfa) and 1996 (follitropin beta). These had superior purity and a more homogenous glycosylation pattern compared with urinary or pituitary FSH. Subsequently recombinant versions of LH and hCG have been developed, and biosimilar versions of follitropin alfa have received marketing authorization. More recent developments include a recombinant FSH produced using a human cell line, and a long-acting FSH preparation. These state of the art products are administered subcutaneously via pen injection devices.

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): Insights into the evolution of thyrotropin receptor specificity

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH–TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T4-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone–receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11–20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin β subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T4, thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.

Recombinant or Urinary Human Chorionic Gonadotropin in Ovulation Induction?

Recombinant or Urinary Human Chorionic Gonadotropin in Ovulation Induction?

Gonadotropin stimulation: past, present and future.

Gonadotropin therapy is so central to infertility treatment that it is easy to overlook the considerable discovery and research that preceded production of the effective and safe products available today. The history underpinning this development spans over 300 years and provides a splendid example of how basic animal experimentation and technological advances have progressed to clinical application. Following the discovery of germ cells in 1677 and realizing, in 1870, that fertilization involved the merging of two cell nuclei, one from the egg and one from sperm, it took another 40 years to discover the interplay between hypothalamus, pituitary and gonads. The potential roles of gonadotropin regulation were discovered in 1927. Gonadotropin, such as pregnant mare serum gonadotropin (PMSG), was first introduced for ovarian stimulation in 1930. However, use of PMSG leads to antibody formation, and had to be withdrawn. Following withdrawal of PMSG, human pituitary gonadotropin (HPG) and urinary menopausal gonadotropin (hMG) appeared on the market, and 50 years ago the first child was delivered by our group in 1961 and opened the path to controlled ovarian stimulation. HPG produced good results, but its use came to an end in the late 1980s when it was linked to the development of Creutzfeldt-Jakob disease (CJD). HMG preparations containing a high percentage of unknown urinary proteins, making quality control almost impossible, were then the only gonadotropins remaining on the market. With the availability of hMG, clomiphene citrate, ergot derivatives, GnRH agonists and antagonists, as well as metformin, algorithms were developed for their optimal utilization and were used for the next four decades. Following the first human IVF baby in 1978 and ICSI in 1991, such procedures became standard practice. The main agents for controlled ovarian stimulation for IVF were gonadotropins and GnRH analogues, with batch to batch consistent gonadotropic preparations; methods could be developed to predict and select the correct dose and the optimal protocol for each patient. We are now seeing the appearance of gonadotropin with sustained action and orally active GnRH analogues as well as orally active molecules capable to stimulate follicle growth and inducing ovulation. These new developments may one day remove the need for the classical gonadotropin in clinical work.

Ovarian stimulation: from basic science to clinical application

Treatment for infertility, including ovarian stimulation, was first introduced almost 100 years ago. At this time, radiation therapy became an established treatment, and it was only some decades later that the problem of radiation-induced cancer emerged. Non-human gonadotrophins, such as pregnant mare serum gonadotrophin (PMSG), and human pituitary gonadotrophins (HPG), were commonly used for hormonal stimulation procedures. However, use of PMSG led to antibody formation, and it was therefore only useful for the first treatment cycle. HPG produced good results, but its use came to an end in the late 1980s when it was linked to the development of Creutzfeldt-Jakob disease. The first hormonal product from human menopausal urine to be used was human menopausal gonadotrophin (HMG), followed later by purified preparations of this product. All of these preparations contained a high percentage of unknown urinary proteins, which interfered with batch-to-batch consistency. This changed with the introduction of recombinant gonadotrophins, produced from an immortalized/standardized mammalian cell line (CHO). More recent developments include the introduction of long-acting gonadotrophin formulations. The development of gonadotrophin-releasing hormone (GnRH) analogues and more recently the use of GnRH antagonists has helped to improve ovarian stimulation protocols by optimizing their efficacy, and making them easier to administer.

Iatrogenic Creutzfeldt–Jakob disease in Australia: time to amend infection control measures for pituitary hormone recipients?

From 1967, the Australian Human Pituitary Hormone Program offered treatment for short stature and infertility using human cadaver-acquired pituitary hormones (human growth hormone [hGH] and human pituitary gonadotrophin [hPG]). The program was suspended in 1985when a growth-hormone recipient in the United States developed Creutzfeldt-Jakob disease (CJD), an incurable and rapidly progressive neurodegenerative disorder. Since this time, recipients have lived with the significant anxiety that they have an elevated risk of developing CJD. Furthermore, additional CJD infection control measures are required when recipients undergo some types of surgery. As it is 20years since the last Australian pituitary hormone recipient developed CJD, we evaluated the risk for Australian recipients of developing iatrogenic CJD, and compared Australian data with data from New Zealand and selected other countries who had pituitary hormone programs. Our evaluation indicates that pituitary hormone recipients in Australia have the lowest risk of developing iatrogenic CJD, and that Australia is the only country not to have experienced ongoing CJD-related deaths. Thus, we believe that: in the Australian hGH recipient cohort, the risk of developing CJD is sufficiently low for this cohort to no longer require additional infection control measures in the health care setting; and in the Australian hPG recipient cohort, if another 5years elapses with no further occurrence of CJD in this group, the hPG recipient cohort could also be considered as not requiring additional infection control measures in the health care setting. These recommendations should not be misunderstood as implying that there is no ongoing risk, but that the risk is acceptably low and generally in keeping with guidelines that stratify the risk.

The follicular phase stimulated by human pituitary gonadotropin and the luteal phase following in vitro fertilization.

The pattern of follicular and luteal phase levels of serum estradiol-17 beta (E2) and progesterone (P) are compared between 5 conception (group P) and 27 nonconception (group N) cycles of patients undergoing in vitro fertilization and embryo transfer. Follicle growth was induced with human pituitary gonadotropin and controlled by daily ovarian ultrasonography measuring the follicular diameters. Group N has significantly (p less than 0.025) higher E2 levels from day -10 up to d-6 as compared with the group P. The day of laparoscopy is designated day 0. In group N the length of E2 phase with E2 levels, exceeding 1.84 nmol/l (500 pg/ml) serum, is greater (p less than 0.001) than in group P 6.9 and 5.6 days, respectively. It is suggested that modifications occur in the endometrium caused by high E2 levels acting for a long time, which disturb the implantation of transferred embryos. No significant differences exist between group N and P, if the diameters of the largest follicle or the mean diameters of the secondary follicles or the number of follicles with a diameter greater than 14 mm are compared. No significant correlations were found between these variables and the E2 levels. A significant correlation (r = 0.85, 14 patients) exists between the E2 rise and the increase of follicular diameters.

A comparative review of ovulation and pregnancy rates by gonadotrophin preparations in women with group 1 who anovulatory infertility

OBJECTIVE: The aim of this study was to compare ovulation and pregnancy rates in a large cohort of women with Type I WHO anovulatory infertility undergoing ovulation induction (OI) with gonadotrophin preparations containing varying amounts of luteinizing hormone (LH). DESIGN: A retrospective study of patients with Type I WHO anovulatory infertility undergoing gonadotrophin OI at our institution between 1981 and 2006. MATERIALS AND METHODS: Gonadotrophin preparations included human pituitary gonadotrophin (hPG), human menopausal gonadotrophin (hMG), purified urinary follicle stimulating hormone (uFSH) and highly purified urinary FSH (hpuFSH), and recombinant FSH (rFSH). All patients were entered into a database (Foxpro). Statistical analysis using SPSS was performed. RESULTS: A total of 223 patients undergoing 731 cycles of OI with gonadotrophins were analyzed. The mean patient age was 30 years (range 19-49). The mean FSH usage per cycle was 1413 units. Overall, ovulation and pregnancy rates per cycle were 81.5% and 22.1% respectively with a cumulative pregnancy rate of 60% per woman. The ovulation rate per cycle was significantly higher with purified and highly purified urinary FSH (90%) and rFSH (89%) compared to hMG (59%) and hPG (41%) (p<0.0001). However, there was no difference in pregnancy rates per ovulatory cycle between the different gonadotrophin preparations. CONCLUSIONS: The belief that LH is crucial for folliculogenesis has become increasingly accepted; in particular, supplementation of LH in hypogonadotrophic women undergoing OI. The use of recombinant FSH, purified and highly purified urinary FSH for OI in this large series of patients with WHO Type I anovulatory infertility, was more effective than other gonadotrophin preparations in achieving ovulation and equally as effective in terms of pregnancy. These findings question the hypothesis that additional LH is required for OI in WHO Type I patients.

Expression and role of VEGF receptors in pituitary adenomas

Vascular endothelial growth factor (VEGF) is known to exert a highly specific mitogenic action on endothelial cells through two tyrosine kinase receptors: VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) which are almost specifically expressed in endothelial cells. However, it has been shown that VEGF receptors may also be expressed by non-endothelial cells, especially by tumor cells. We have recently shown that pituitary adenoma cells produce variable amounts of VEGF and have now started to study the expression of VEGF receptors in normal human pituitary and pituitary adenomas by RT-PCR, in situ hybridisation and immunohistochemistry. Expression of VEGFR1 and VEGFR2 mRNA was found in normal pituitary and in the 49 adenomas (26 nonfunctioning, 9 somatotroph, 8 corticotroph and 6 lactotroph tumors) studied so far. The expression was variable and no correlation between VEGFR1 expression and tumor grade and vessels number was found. In normal pituitary, VEGFR1 immunoreactivity was observed to co-localize with ACTH-, FSH-, GH- and LH-secreting cells, but not in endothelial cells, suggesting that VEGF may affect function of endocrine cell types by paracrine mechanisms. In contrast, VEGFR2 immunoreactivity was detected only in the vascular formations, in both normal pituitary and pituitary adenomas. Immunohistochemical analysis for VEGFR2 in 33 pituitary adenomas (9 nonfunctioning, 7 somatotrophs, 6 corticotrophs and 11 lactotrophs) revealed immunoreactivity in the vascular formation of 18 tumors and we observed that somatotroph and corticotroph adenomas showed the highest number of cases with VEGFR2 immunoreactivity on vessels. However, we found no correlation between VEGFR2 expression and extent of vascularization. We have then observed that HP75, a human non-functioning pituitary adenoma cell line secreting gonadotropins, expresses both VEGFR-1 and –2 mRNA and human VEGF165 is able to stimulate cells proliferation in a dose-dependent manner. This finding could suggest again a possible paracrine/autocrine action of VEGF on endocrine and tumor cells.

Ovarian stimulation: from basic science to clinical application

Treatment for infertility, including ovarian stimulation, was first introduced almost 100 years ago. At this time, radiation therapy became an established treatment, and it was only some decades later that the problem of radiation-induced cancer emerged. Non-human gonadotrophins, such as pregnant mare serum gonadotrophin (PMSG), and human pituitary gonadotrophins (HPG), were commonly used for hormonal stimulation procedures. However, use of PMSG led to antibody formation, and it was therefore only useful for the first treatment cycle. HPG produced good results, but its use came to an end in the late 1980s when it was linked to the development of Creutzfeldt–Jakob disease. The first hormonal product from human menopausal urine to be used was human menopausal gonadotrophin (HMG), followed later by purified preparations of this product. All of these preparations contained a high percentage of unknown urinary proteins, which interfered with batch-to-batch consistency. This changed with the introduction of recombinant gonadotrophins, produced from an immortalized/standardized mammalian cell line (CHO). More recent developments include the introduction of long-acting gonadotrophin formulations. The development of gonadotrophin-releasing hormone (GnRH) analogues and more recently the use of GnRH antagonists has helped to improve ovarian stimulation protocols by optimizing their efficacy, and making them easier to administer.

Isolation and characterization of human pituitary chorionic gonadotropin.

Human CG (hCG) is the hormone associated with the maintenance of pregnancy. Although three related glycoprotein hormones, LH, hFSH, and hTSH, are secreted by the pituitary, HCG is the only one of this family of glycoprotein hormones that is produced by the placenta in primates to maintain the steroid hormone secretions of the corpus luteum. Although hCG is not considered to be a pituitary hormone, hCG-like immunoreactive materials have been reported in pituitary tissue, blood, and urine from healthy nonpregnant individuals for 2 decades, but it was never isolated. We now report the purification and characterization of pituitary hCG from acetone-preserved human pituitary glands. After gell permeation chromatography, the fractions containing hCG molecules were pooled and purified by immunoaffinity chromatography using antibodies to the COOH-terminal region of hCGbeta. Amino acid analyses, amino-terminal sequence analyses, as well as mass spectrometric studies gave similar results for both pituitary hCG and hCG purified from the urine of pregnant women. Analyses for sulfate and sialic acid contents demonstrated that pituitary hCG contained both sulfate and sialic acid. In vitro biological activity of pituitary hCG indicated that it was 50% as active as hCG purified from the urine of pregnant women in cAMP assays.

Isolation and characterization of human pituitary chorionic gonadotropin

Isolation and characterization of human pituitary chorionic gonadotropin

Pregnancy in women with Kallmann’s syndrome

To induce of ovulation and pregnancy in women with Kallmann's syndrome. Retrospective study. Three women with hypogonadotropic hypogonadism and anosmia with a desire for pregnancy. Investigation of hypothalamic-pituitary-ovarian function and induction of ovulation by pulsatile GnRH or intramuscular human pituitary gonadotropins (hPG) or hMG with hCG. Successful induction of ovulation as measured by serum P levels and successful pregnancy. Ovulation was induced successfully in all three patients on more than one occasion and nine pregnancies occurred. Gonadotropin-releasing hormone was given IV by an electronically timed syringe driver. A total of 12 pulsatile GnRH cycles resulted in two pregnancies, 6 of these cycles being in one patient who did not ovulate or conceive with this therapy. Ovulation occurred in 10 of 16 hMG or hPG cycles, with conception in 7 of these. Gonadotropin usage was higher in these women compared with women with hypogonadotropic hypogonadism without anosmia (2,850 compared with 2,100 IU per treatment cycle), and the follicular phase was longer. All three women conceived and had children after induction of ovulation. The success rate of these therapies in Kallmann's syndrome appears to be high in spite of very few reports in the literature.

Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid ( N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80°C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard ( N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5–100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5–13.7 mol Neu5AC/mol FSH, whilst a range of 1.1–9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoform was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.

Inhibition of human hepatocarcinoma cell proliferation by mammalian and fish gonadotropin-releasing hormones.

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

Inhibition of human hepatocarcinoma cell proliferation by mammalian and fish gonadotropin-releasing hormones

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

Folliculogenesis--the stimulated way.

The initial techniques of stimulating follicular development in the anovulatory woman involved the use of human pituitary gonadotrophin (hPG) and thus replaced the function of the pituitary gland. Despite extreme care with administration of hPG and extensive monitoring to assess the ovarian response, ovarian hyperstimulation and multiple pregnancy were common. Less expensive and easier methods of treatment soon followed with oral clomiphene citrate (early 1960s), oral bromocriptine (early 1970s) and pulsatile gonadotrophin-releasing hormone (late 1970s) being used. Currently all of these methods, alone or in combination, are employed and successful ovulation induction (except in women with elevated FSH levels) can now virtually be guaranteed. Controlled ovarian hyperstimulation, just the outcome one was attempting to avoid in the treatment of anovulatory women, has become the treatment of choice for women having in vitro fertilization (IVF) or gamete intrafallopian transfer (GIFT). The extra oocytes produced by this treatment results in more embryos being available for transfer and/or freezing and improves the overall pregnancy rate. The concurrent use of gonadotrophin-releasing hormone agonists (GnRH-a) has resulted in more mature oocytes being developed, less cancelled cycles for a spontaneous midcycle LH surge, and allowed even more embryos to be produced thereby increasing the pregnancy rate further to the current expected 20% per cycle commenced. As techniques are further modified, adverse effects of elevated LH levels on pregnancy and take home baby rates should be able to be overcome, and oocyte freezing and long-term storage should become a possibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.

Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.