Current Pharmaceutical Biotechnology - Volume 13, Issue 3, 2012

Infertility, or the inability to conceive, is a problem affecting millions of couples around the world. In many cases, the infertility can be treated effectively only by means of assisted reproductive techniques (ART). Ovarian stimulation is a key component of ART and is directed towards the ultimate goal of obtaining an adequate number of good quality oocytes while preventing excessive ovarian response. Recent years have witnessed a significant expansion of the pharmacological armamentarium useful in ovarian stimulation protocols. In this issue Current Pharmaceutical Biotechnology a wide spectrum of relevant and fascinating topics in the area of ovarian stimulation are analyzed and discussed by a panel of distinct international authors. In the first article of this series, De Leo and co-workers offer a comprehensive review on the pharmacological aspects and different clinical applications of recombinant gonadotropins in the treatment of infertility in all its aspects. The manuscript from Fatemi et al. give a full analysis of the pharmacological agents available for ART programs. New hope and challenges in the area are also illustrated. The outcome of ART is largely influenced by the ovarian reserve, i.e. the capacity of the ovary to provide eggs that are capable of fertilization. La Marca and associates evaluate the current literature on ovarian reserve testing. The potential role of LH supplementation in ovarian stimulation remains controversial. The manuscript from Munoz and associates, besides discussing the issues surrounding the use of this gonadotropin in ovarian stimulation, focuses on the identification of patient subpopulations that may benefit from LH supplementation. Pharmacogenetics deals with the study of the relationship between individual gene variants and variable drug effects. The manuscript from Loutradis et al., reviews evidences linking gene polymorphism and ovarian response to gonadotropin stimulation. The potential clinical role of pharmacogenetic, especially in predicting ovarian response, is also outlined. In a related research paper presented by Anagnostou et al., the occurrence of differences in ovarian stimulation outcome and pregnancy rates among patients with different genotype combination profiles is presented. McCulloh and associates provide in their manuscripts a clear and novel description of how FSH levels change in blood during gonadotropin administration and provide understanding of how the underlying controllers affect FSH levels, and provide methods for administration that may improve the results of gonadotropin administration. The topic of progesterone elevation during ovarian stimulation, and its possible negative impact on ART outcome is addressed by a meta-analysis by Kolibianakis et al., showing that progesterone elevation on the day of hCG administration reduces the probability of clinical pregnancy in patients treated with GnRH antagonists and gonadotropins. It has been an honor working with the authors and editing this journal issue. I wish to acknowledge the referees for their conscientious efforts and constructive remarks.

Pharmacological ovarian stimulation has a major role in reproductive medicine and has been used in anovulatory patients and in the induction of multifollicular development required for the procedures of assisted reproductive techniques (ART). Currently, gonadotropins are the most important tools to proceed with ovarian stimulation for all purposes, including ART and anovulation disorders, like hypogonadotropic hypogonadism and hypothalamic hypophyseal dysfunction. Gonadotropin preparations derived from human urine have been used clinically since the early 1960s and the first urine-derived preparation containing only FSH (urofollitropin) became available in 1983. More recently, the application of recombinant DNA technology has resulted in the development of recombinant FSH produced in mammalian cells. In the last period, LH became available by recombinant DNA technology and is now a new option for protocols of ovarian stimulation. Treatment with gonadotropins has been shown to be effective in males affected by hypogonadotropic hypogonadism. This success has resulted in attempts to utilize FSH therapy in oligozoospermic men, aimed at obtaining a quantitative increase in sperm count. The purpose of this review was to examine the pharmacological aspects and different clinical applications of recombinant gonadotropins (FSH, LH, hCG) in the treatment of female infertility in all its aspects and their use also in the treatment of male infertility. This review will trace these events, from the past through to the present, and conclude with a glance towards the future.

In assisted reproductive technology, medications and ovarian stimulation play a crucial role. The availability of gonadotrophins and GnRH analogues has allowed the tailoring of several stimulation schemes. The two most commonly used gonadotrophin forms are urinary hMG and recombinant FSH in combination with GnRH agonists or GnRH antagonists. Cycles stimulate with recombinant FSH appear to have a higher risk of premature progesterone rise in the late follicular phase, if not triggered on time. Recently, corifollitropin alfa, a new long acting recombinant FSH was introduced which sustain multiple follicular growth for 7 days in women undergoing ovarian stimulation using GnRH antagonists. GnRH antagonist and agonist do have similar live birthrate. However, GnRH antagonists reduce significantly the risk of OHSS. Moreover, with the introduction of GnRH antagonists, it became possible to trigger ovulation with a bolus of a GnRH agonist as an alternative to hCG. The early OHSS is eliminated completely with the GnRH agonist trigger. However, due to an uncorrectable luteal phase deficiency, a poor clinical outcome is present, when GnRHa is used to trigger final oocyte maturation in IVF/ICSI antagonist protocols. Therefore, it has been suggested to cryopreserve the embryos and transfer in consecutive natural cycles. Future trials should aim to eliminate OHSS and multiple pregnancy rates by performing a single stimulation in a simplified corifolitropin alfa/GnRH antagonist cycle triggered by a GnRH agonist followed by Cryo-thawed SET in consecutive natural cycles. With this approach, the two major complications of COH for IVF could be eliminated without jeopardizing the outcome.

Markers of ovarian reserve are associated with ovarian aging as they decline with chronologic age, and hence may predict stages of reproductive aging including the menopause transition. Assessment of ovarian reserve include measurement of serum follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH), and inhibin-B. Ultrasound determination of antral follicle count (AFC), ovarian vascularity and ovarian volume also can have a role. The clomiphene citrate challenge test (CCCT), exogenous FSH ovarian reserve test (EFORT), and GnRH-agonist stimulation test (GAST) are dynamic methods that have been used in the past to assess ovarian reserve. In infertile women, ovarian reserve markers can be used to predict low and high oocyte yield and treatment failure in women undergoing in vitro fertilization. However the markers may have limitations when an in depth analysis of their accuracy, cost, convenience, and utility is performed. As ovarian reserve markers may permit the identification of both the extremes of ovarian stimulation, a possible role for their measurement may be in the individualization of treatment strategies in order to reduce the clinical risk of ART along with optimized treatment burden. It is fundamental to clarify the cost/benefit of its use in the ovarian reserve testing before initiation of an IVF cycle and whether the ovarian reserve markers-determined strategy of ovarian stimulation for assisted conception may be associated to improved live birth rate.

LH is a glycoprotein that plays a crucial role in folliculogenesis during the natural ovarian cycles. It has the same activity and shares receptors with hCG. However the use of LH in combination with FSH in controlled ovarian stimulation remains controversial. A practical approach concerning the usefulness of LH according to the endogenous level of LH is described herein. Specific groups of patients can benefit from ovarian stimulation with LH. New applications of LH/hCG activity are also discussed.

Obtaining an adequate number of good quality oocytes while minimizing adverse drug reactions (ADRs) and cycle cancellation rates is considered the gold standard in controlled ovarian hyperstimulation (COH) for fertility treatment. Patients who undergo IVF/ICSI cycles tend to present with different responses to exogenous gonadotrophin administration. Research has shown that the secret probably lies in the various single nucleotide polymorhisms (SNPs) in their receptor genes. The decryption of human genome provided specialists with additional information in assessing and even predicting ovarian response to COH. In this context, the study of Pharmacogenomics, Pharmacogenetics and SNPs unravels as a promising field in optimizing fertility treatment. Several SNPs in FSH and estrogen receptor genes have been detected so far, but only three of them, one in FSH receptor and two in estrogen receptor genes have been associated with ovarian response to COH. It seems that the Asn/Ser variant of the FSH receptor functions more efficiently, while the Ser/Ser and Asn/Asn variants have a tendency to resist to FSH stimulation. With regards to estrogen receptor 1 (ESR1), the Pvull and the Xbal polymorphisms seem to be associated with differences in the response to ovarian stimulation, while the Rsal polymorphism in estrogen receptor 2 (ESR2) is currently under investigation. There exists evidence supporting the hypothesis that a set of genes, all related to the FSH hormone mechanism of action, may participate along with other factors to the control of ovarian response to FSH, thus a cautious interpretation of polymorphism detection results is considered mandatory. However, identifying potential genetic markers that could predict ovarian response and implementing them in routine screening tests for every woman entering an IVF/ICSI cycle, would be able to tailor fertility treatment to each patients needs thus maximizing the success rate and eliminating potential side-effects of fertility drugs.

Purpose: Previous studies in humans concluded that a multigenic model including specific FSHR, ESR1 and ESR2 genotype patterns may partially explain the poor response to FSH. The aim of our study is to analyse three different loci -polymorphisms in ESR1 Pvu II, ESR2 Rsa I and Ser680Asn FSH receptor gene- in a Greek population and their involvement in stimulation outcome and pregnancy rates. Methods: Each locus was studied alone, and in combination with the others. We performed both restriction fragment length polymorphism analysis and real-time polymerase chain reaction. A total of 109 normally ovulating female patients underwent IVF or ICSI. Results: Studying each locus alone, no significant results were drawn for ESR1 and ESR2 genes. Concerning the FSHR polymorphism, the women carrying the AA variant presented higher total amount of gonadotrophins used (P=0,048) and tended to have higher number of stimulation days (P=0,057). Considering the ESR1 and FSHR gene polymorphisms in combination, the TC/SA combination presents the highest number of pregnancies in poor responders group (3/4 pregnancies carried this genotype), in good responders group (4/12 pregnancies carried this genotype) and in the total population (10/26 pregnancies carried this genotype). Except the CC/AA combination, all other genotype combinations presented incidence of pregnancy, with TC/SA having the highest incidence. The CC/AA genotype presents the worst profile of ovulation induction, confirming a poor responder profile: the total amount of gonadotrophins used was highest in CC/AA group (P<0,05). The peak E2, the number of follicles and of retrieved oocytes and the pregnancy rate were significantly lower (P<0,05). This genotype combination seems to be over-presented in the poor responders group in a statistically significant way (P=0,038). Women with CC/AA combination have 1,5-2,4 times more risk to be poor responders in comparison with women that do not carry that combination. Conclusion: This study supports the hypothesis that a multigenic model, including the well studied ESR1 and FSHR genes is involved in the controlled ovarian stimulation outcome indicating that the CC/AA genotype presents the worst ovulation induction profile, while the TC/SA genotype presents the higher number of pregnancies in our population.

Background: Despite the dangers of gonadotropin administration, our understanding of serum levels of gonadotropins in our patients is poor. We created a mathematical model of gonadotropin administration in order to learn about the temporal profiles of Follicle Stimulating Hormone (FSH) levels in patients related to the doses of different gonadotropin products administered. Methods: Model parameters for each product were determined from published pharmacokinetic information available from the manufacturers. Results: For each product, serum levels of FSH rose then fell following a single injection of FSH. When a gonadotropin dose was injected repeatedly (daily), each injection resulted in a peak level of FSH that was higher than the level attained from the previous injection. Daily FSH peaks rose asymptotically, requiring 3-10 days to approach the maximum. The maximum averaged 3.5 times the peak of the first injection. Decline of FSH following the final injection required 3-10 days to approach the baseline. Time courses and peak values of FSH using different gonadotropin preparations varied widely. Conclusion: Knowledge of the slow changes of FSH that occur during modeled FSH administration will provide a framework for understanding the gonadotropin profiles that occur in patients during controlled ovarian hyperstimulation.

Background: Serum levels of follicle stimulating hormone (FSH) in patients change slowly during repeated daily injections of gonadotropin, requiring several days to achieve stable values. Using a numerical (computer) model we mimicked the serum levels of FSH using two kinetic constants that describe biological phenomena: diffusion of FSH from the injection site into the blood (τin) and disappearance of FSH from the blood by excretion/degradation (τout). We investigated the effects of these two parameters on the FSH profiles seen in the model. Methods: The parameters, τin and τout, were systematically changed to determine their effects on the delay from injection to the peak following a single injection (Tmax). Other values were investigated systematically during repeated daily injections. Results: The model's parameters τin and tout affected Tmax and several features of FSH level during daily injections of gonadotropin. These included the maximum level of FSH in the serum (relative to the level attained following the first injection), the rate of rise of peaks following injections after the first, and the shortening of the delay from injection to peak. Conclusion: Knowledge of τin and τout permits us to predict serum FSH levels and time course during daily injections of gonadotropin.

Background: A numerical model with two pharmacokinetic parameters (the rate of diffusion into the blood (τin) and the rate of disappearance from the blood (τout)) mimicks the slow changes of follicle stimulating hormone (FSH) seen in serum. It is often desired to change serum FSH levels more rapidly. Since the pharmacokinetic parameters cannot be changed in patients, we used the model to investigate changing the amount and timing of gonadotropin administration to change FSH levels in serum more rapidly. Methods: Parameters defining the model were obtained as described previously [1]. The doses administered, and the timing of administration were individually specified. Results: The rise of serum FSH was made more rapid by administering an initial injection larger than the subsequent injections. When a rapid decrease of serum FSH was desired, administration of the new, lower dose was delayed. Conclusion: Use of a model that simulates the serum levels of FSH during gonadotropin administration provided a framework for learning how to achieve the desired serum FSH levels more rapidly in patients. With knowledge of a particular patient's τin and τout, optimal FSH administration for that patient can be determined through modeling.

The current meta-analysis aimed to answer the following research question: is progesterone elevation on the day of hCG administration associated with the probability of clinical pregnancy in women undergoing ovarian stimulation for IVF using GnRH antagonists? A literature search in MEDLINE, EMBASE and CENTRAL electronic databases followed by extensive hand-searching from two independent reviewers was performed to identify relevant studies. Eventually five eligible studies (n=585 patients) were identified. No significant differences were present between patients with and those without progesterone elevation regarding female age, duration of stimulation and total dose of gonadotrophins required. However, patients with progesterone elevation were characterized by higher serum estradiol levels on the day of hCG administration (+956 pg/ml, 95% +248 to +1664, random effects model, p=0.008) and more COCs retrieved (+2.9, 95% CI +1.5 to +4.4, fixed effects model, p<0.001). Progesterone elevation on the day of hCG administration was associated with a significantly decreased probability of clinical pregnancy per cycle (-9%, 95% CI -17 to -2, fixed model effects, p&equals0.02). In conclusion, in patients treated with GnRH antagonists and gonadotrophins, progesterone elevation on the day of hCG administration is significantly associated with a lower probability of clinical pregnancy.

Freeze drying, or lyophilization is widely used for biopharmaceuticals to improve the long term storage stability of labile molecules. This review examines general theory and practice of rational lyophilization of biopharmaceuticals. Formulation development involving the selection of appropriate excipients, their associated physical properties, and mechanism of action in achieving a stable drug product are primary considerations for a successful lyophilization program. There are several parameters considered critical on the basis of their relationship to lyophilization cycle development and protein product stability. This along with the importance of analytical methods to provide insight toward understanding properties of drug product stability and cake structure are discussed. Also, aspects of instability found in lyophilized biopharmaceutical products, their degradation pathways and control are elucidated. Finally, container-closure requirements and drug product handling are described in context of the caveats to avoid compromising drug product quality.