Gonadotropin releasing hormone analogs as part of controlled ovarian stimulation

I describe how we identified the need to block the luteinizing hormone (LH) surge when trying to control the processes of luteinization and ovulation within the clinic. The first step, in fact, was using ovarian ultrasound evaluation of follicular development in the natural cycle (published in 1979) and then when the ovary was stimulated with exogenous follicle stimulating hormone. We observed that induced multiple follicular development often led to “premature” LH surges—which occurred before the leading follicle had achieved normal preovulatory dimensions. The work required both ovarian ultrasound and reliable radioimmunoassays, which were not always available. When early work with gonadotropin releasing hormone agonists showed that they could suppress LH activity, it was the logical step to try to use them to perform that task during the induction of multiple follicular development. High frequency administration of the gonadotropin releasing hormone-agonist successfully achieved sustained LH suppression through the follicular phase allowing clinical control of luteinization and ovulation. I describe how we identified the need to block the luteinizing hormone (LH) surge when trying to control the processes of luteinization and ovulation within the clinic. The first step, in fact, was using ovarian ultrasound evaluation of follicular development in the natural cycle (published in 1979) and then when the ovary was stimulated with exogenous follicle stimulating hormone. We observed that induced multiple follicular development often led to “premature” LH surges—which occurred before the leading follicle had achieved normal preovulatory dimensions. The work required both ovarian ultrasound and reliable radioimmunoassays, which were not always available. When early work with gonadotropin releasing hormone agonists showed that they could suppress LH activity, it was the logical step to try to use them to perform that task during the induction of multiple follicular development. High frequency administration of the gonadotropin releasing hormone-agonist successfully achieved sustained LH suppression through the follicular phase allowing clinical control of luteinization and ovulation. The year 1977 is a landmark for the understanding of mammalian reproduction because of the Nobel prize for Andrew Schally, with his colleague Roger Guillemin, being awarded for his remarkable endeavors and achievements with gonadotropin releasing hormone (GnRH) and its analogs. However, it is often overlooked that they shared the honor on that day with Rosalyn Yalow, who was awarded it “for the development of radioimmunoassays of peptide hormones.” This latter contribution was key to the establishment of the singular professional sub-specialty of “reproductive endocrinology” simply because her work led to our ability to measure hormones at extremely low concentrations in small amounts of biological fluids. This was a revolutionary step, and it is where my contribution began. The year before their joint recognition (1976), I moved to Scotland to join a small team working on infertility among women with normal menstrual rhythm to carry out postdoctoral work in a dedicated laboratory associated with the university department of gynecology. The concept (funded by the Scottish government) required me to set up assays and measure steroid and gonadotrophin hormones in women under various conditions. The “dedicated” reproductive endocrinology laboratory was located in a rather unprepossessing part of the Royal Infirmary on the east side of Glasgow. The building itself dated from 1794 and had seen little investment over the following 200 years, except for some beautiful fume cupboards constructed using polished oak bearing some exotic and imaginative graffiti. The team was led by the remarkable Ian Coutts, with whom I enjoyed many years of work and fun: although, despite many attempts, I could never quite match his lifelong commitment to Scotland’s main export. For many years now, immunoassays have deployed monoclonal antibodies with high specificity, often targeted at specific parts of molecules with great precision, and able to operate with high degrees of sensitivity. Although a great step forward, the original radioimmunoassays deployed polyclonal antibodies from various mammalian serum sources, and the characteristics of these amazing tools were complex, with each iteration having unique characteristics, and they could also vary between batches of the same anti-serum. This was particularly important for the larger peptide hormones, including the gonadotrophins. Most luteinizing hormone (LH) assays were characterized or compromised by high degrees of cross-reaction with human chorionic gonadotropin (HCG) and/or free alpha sub-unit of the gonadotropins. The former can be present in high concentrations, so even a low degree of cross-reaction can lead to confusing measurement values. The alpha sub-unit, common to all gonadotrophins and thyroid stimulating hormone, is rarely present in free form in high concentrations, but there are specific circumstances when they can contribute to confusing observations. Figure 1 shows what happens after starting GnRH-agonist treatment when measured using assays focused on the whole LH, focused mainly on the beta-subunit, compared with one focused on the alpha-subunit alone. It is easy to see how confusing profiles can be experienced when the assay antibody may give undue weight to the alpha sub-unit when measuring what is thought to be LH. This can lead to difficulties in interpretation and undermine confidence in the results. The steroid hormones also showed cross-reactions with other steroids of similar size and complexity and progesterone assays with other C21 steroids and corticosteroids, which can be present in significantly higher concentrations. This means that even if there is a low degree of cross-reaction, the effect on the results can be significant. These complexities meant that the interpretation of apparent results could be confusing, and we were always seeking antibodies with properties that suited our tasks better. Ian Coutts was a well-connected individual, and he had a remarkable ability to “find” people who had antibodies that they were willing to provide from many and various sources and also to negotiate “reasonable” terms of purchase: some rather unconventional. It was my role to assess the validity and functionality of the tests for the hormones, or other analytes, we planned to measure. My work began with a proposal to define normal reproductive hormone profiles throughout the menstrual cycle and explore differences in women suffering from a minimum of 3 years of infertility. These same infertile women were then to be stimulated with modest doses of urinary human menopausal gonadotropins (HMG) in the early follicular phase, with responses monitored by daily serum samples analyzed for the same hormones. Although this strategy includes their own control data, we could not conclude that the data we were to collect represented “normal.” It was a matter of great fortune that a young German doctor named Jochen Hackeloer contacted us to assert that he thought he could identify follicles growing in the ovary using the ultrasound equipment in Ian Donald’s department on the west side of the city. I have to admit that the picture he showed me looked like a poor quality representation of the lunar surface rather than a follicle! So, we set up a project in which he would measure the size of follicles (follicle diameter [FD]) in a series of volunteers, and we would measure the corresponding estradiol (E2) concentrations. He formed the “ovary club” of numerous remarkable volunteers, who provided his scan data and serum samples: this meant that we had our blood samples from “normal” volunteers, so I was very pleased that he had come to us. Come the study day of reckoning, when the E2 measurements were correlated with Jochen’s FD estimates, we were profoundly disappointed that there was no correlation between the 2 primary measurements. However, I had also used the samples to determine normal values of LH and progesterone, using the best antibodies I had tested thus far. The others were unaware that I had these data, so when I suggested that we could examine the follicular phase data in isolation, I was suddenly very popular. Later that day, when we rearranged the data by reference to the LH peak, the data transformed magically into a perfect correlation between the FD and E2. I think of this as a critical moment in my personal history, and the work was soon reported in the world's first demonstration of ultrasound measurement of ovarian follicles published in 1979, causing international interest (1Hackeloer B.J. Fleming R. Robinson H.P. Adam A.H. Coutts J.R. Correlation of ultrasonic and endocrinological assessment of follicular development.Am J Obstet Gynecol. 1979; 135: 122-128Abstract Full Text PDF PubMed Google Scholar). The rearrangement of data meant that the measurements of the corpora lutea and the luteal phase samples were removed from the dataset. Overall, it meant that we could be satisfied that our LH, E2, and progesterone assays were reliable at physiological concentrations, and we suddenly had a new tool by which to explore ovarian function. This new tool gave us a novel and unexpected aspect to examine, both in questions regarding normality and under stimulation. Critically for future events, we noted that the size of the periovulatory follicle in the normal cycle on the day of the LH surge was between 18 and 26 mm in diameter, with a mean of 22 mm. Correspondingly, we concluded that a FD of 20 mm should be set as a target size range for the lead follicle during ovarian stimulation. Jochen Hackeloer departed to further his career, but other young researchers were keen to follow up his work with us, as we added FD data to the resources in the study of the effects of early follicular phase HMG treatment in women with normal menstrual rhythm. It meant that we could monitor follicular growth simultaneously with an assessment of estrogen output. The initial observations were not as simple as we hoped, as the maximum FD seen at the time of the LH surge was often much less than in the normal cycle. Figure 2 shows an example of the effect of HMG injections on days 1, 3, and 5 on estradiol, LH, and progesterone: the demonstration of premature luteinization. The “premature” adjective derived from the fact that the follicles were so much smaller than normal at the time of the LH surge compared with that in the normal cycle (in this case, 14mm vs.22 mm in the normal cycle). We concluded that the premature LH surge was a frequent phenomenon under ovarian stimulation and that, correspondingly, we did not have clinical control of either the ovulatory step or oocyte maturation. The work was presented in an abstract form in 1979 and 1980 and received little attention at the time. We were not alone in making the observation and conclusion, as Carl Gemzell and colleagues also reported the phenomenon in 1982, having presented evidence in the abstract form before that (2Gemzell C.A. Kemman E. Jones J.R. Premature ovulation during administration of human menopausal gonadotropins in non-ovulatory women.Infertility. 1978; 1: 1-10Google Scholar). It was clear to us already that if we wanted to have clinical control of the timing of ovulation and egg maturation, we needed a means of blocking the LH surge. With perfect timing, Hamish Fraser, in our neighboring capital city of Edinburgh, reported that treatment of macaques with an agonistic analog of GnRH effectively suppressed ovulation through its ability to suppress LH activity. This work was eventually published in 1980 (3Fraser H.M. Laird N.C. Blakeley D.M. Decreased pituitary responsiveness and inhibition of the luteinizing hormone surge and ovulation in the stumptailed monkey (Macaca arctoides) by chronic treatment with an agonist of luteinizing hormone-releasing hormone.Endocrinology. 1980; 106: 452-457Crossref PubMed Scopus (48) Google Scholar), but his earlier abstracted data had provided the evidence we needed to attempt to use the same GnRH-analog to block the LH surge during ovarian stimulation in women. The work in Sweden with the agonistic analog of GnRH demonstrating its potential role as a contraceptive provided a further indication of its possible role in our setting (4Bergquist C. Nillius S.J. Wide L. Inhibition of ovulation in women by intranasal treatment with luteinizing hormone-releasing hormone agonist.Contraception. 1979; 19: 497-506Abstract Full Text PDF PubMed Scopus (49) Google Scholar). The rights to the use of this drug had been taken up by Hoechst AG, a German chemicals company moving into the world of life sciences. Persuading them that we thought we had specific potential use for their drug was not simple, probably because they thought its potential role was to promote gonadotrophin output rather than exploit its paradoxical down-regulatory effect. However, in the end, they did provide us with some product for initial experiments. The original product (referred to as “Hoe766”, eventually becoming “Buserelin”) was supplied as a nasal spray, administering 100 μg per spray. The short half-life of Hoe766 meant that we needed a high frequency administration over a protracted period to ensure LH surge suppression, but we did not know how long would be required. We initially explored the effect of 5 × 100 μg and 3 × 100 μg sprays over the waking hours. Figure 3 shows the effect on LH of the flare effect under the 2 regimes and the immediate response to nasal spray administration depending on the day of treatment under the “5-sniff” protocol. After a week of treatment, the absolute LH concentration was within the low-normal range, and the immediate response to the application had become negligible. The “3-sniff” protocol was less reliable, and at no stage did we detect complete elimination of the short-term response. The “flare” effect of treatment initiation lasted approximately 3 days. It dictated that we should start treatment in the luteal phase so that the flare effect would influence only luteal secretions. Exogenous follicle stimulating hormone (FSH) administration could be started after menstruation, when pituitary responsiveness should be greatly diminished, within a pharmacologically induced hypogonadotropic environment. Our first use of the GnRH-analog in the luteal phase led to an interesting observation that caused a mild diversion from which I personally learned much. The observation was that the “flare” effect after treatment initiation was luteotrophic: increasing progesterone concentrations—which contrasted with early reports saying that it was luteolytic (5Fleming R. Coutts J.R. LHRH analogues can be luteotrophic.Clin Endocrinol. 1982; 17: 593-599Crossref PubMed Scopus (4) Google Scholar, 6Clayton R.N. Harwood J.P. Catt K.J. hormone to luteal and progesterone 1979; PubMed Scopus Google Scholar). This caused some at the time among people with higher profiles than our and some between us could be as when some to the the measurements of LH showed no concentration that their assay was for the task and interpretation and The initial on the effects of GnRH-agonist administration were in the of follicular so it to be that the LH surge could be in the of high estrogen concentrations experienced under treatment with exogenous gonadotropins. 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