The management of infertility associated with polycystic ovary syndrome

Polycystic ovary syndrome [PCOS] is the commonest cause of anovulatory infertility. Treatment modes available are numerous mainly relying on ovarian stimulation with FSH, a reduction in insulin concentrations and a decrease in LH levels as the basis of the therapeutic principles. Clomiphene citrate is still the first line treatment and if unsuccessful is usually followed by direct FSH stimulation. This should be given in a low dose protocol, essential to avoid the otherwise prevalent complications of ovarian hyperstimulation syndrome and multiple pregnancies. The addition of a GnRH agonists, while very useful during IVF/ET, adds little to ovulation induction success whereas the position of GnRH antagonists is not yet clear. Hyperinsulinemia is the commonest contributor to the state of anovulation and its reduction, by weight loss or insulin sensitizing agents such as metformin, will alone often restore ovulation or will improve results when used in combination with other agents. Laparoscopic ovarian drilling is proving equally as successful as FSH for the induction of ovulation, particularly in thin patients with high LH concentrations. Aromatase inhibitors are presently being examined and may replace clomiphene in the future. When all else has failed, IVF/ET produces excellent results. In conclusion, there are very few women suffering from anovulatory infertility associated with PCOS who cannot be successfully treated today.     

Improving ovarian stimulation protocols for IVF in baboons: lessons from humans and rhesus monkeys

The aim of this review paper is to provide a scientific basis for the development of ovarian stimulation (OS) protocols for in vitro fertilization (IVF) in baboons. Firstly, the evidence available regarding OS for assisted reproduction in baboons is reviewed based on available published data, assessed by a Pub Med search of papers published between 1970 and 2008 using the following key words: baboon, assisted reproduction, IVF, embryo, oocyte. Secondly, we discuss how state-of-the-art or potentially new OS protocols used in humans and in rhesus monkeys may offer guidance for the development of standardized and reliable OS protocols for IVF in baboons. Based on this review and discussion, we conclude that more randomized trials are needed to improve standardization of OS protocols for IVF in baboons with respect to gonadotrophin type, dose, duration of stimulation, ultrasound monitoring, and time interval between ovulation trigger and oocyte retrieval.     

Induction of follicular development and ovulation in seasonally acyclic mares using gonadotrophin-releasing hormones and progesterone.

Deeply acyclic (seasonally anovulatory) mares were treated with GnRH or a GnRH analogue to induce follicular development and ovulation. Courses of GnRH (3--4) were administered at approximately 10-day intervals to reproduce the gonadotrophin surges which precede ovulation in the normal cycle. Exogenous progesterone was administered in an attempt to reproduce the luteal phase pattern. Induced serum FSH concentrations were comparable to those causing follicular development in the normal cycle, but induced LH levels were lower and of shorter duration than those of the periovulatory surge. Three of 4 mares treated with GnRH appeared to ovulate, but did not establish CL. Nine of 10 mares given GnRH analogue also developed follicles during the final treatment course, as did mares treated with progesterone only, while only 1 of 5 untreated control mares showed any ovarian development. Failure to induce final follicular maturation and CL development by this treatment regimen may be due to an inadequate LH surge at the time of the expected ovulation associated with the low preovulatory oestradiol-17 beta surge, possibly caused by the preceding FSH stimulation being inadequate or inappropriate. Progesterone treatment increased baseline FSH concentrations in GnRH-treated mares, and also stimulated follicular development in mares not treated with GnRH, indicating a possible role for progesterone in folliculogenesis and, indirectly, ovulation.     

Gonadotropin releasing hormone: regulation of phospholipid turnover and prostaglandin production in ovarian granulosa cells

The direct effect of gonadotropin releasing hormone (GnRH) upon ovarian function, is initiated by a rapid receptor-mediated increase in phosphatidylinositol (PI) turnover (approximately 5 min) followed by prostaglandin E (PGE, 120 min) and progesterone (120 min) formation, oocyte maturation and induction of ovulation. In contrast, luteinizing hormone (LH) stimulation of oocyte maturation and induction of ovulation is mediated by increased adenosine 3',5'-monophosphate (cAMP, 15 min), progesterone (30 min) and PGE (180 min) production. Both LH and GnRH stimulation of oocyte maturation are inhibited by dibutyryl cAMP and 3-isobutyl-1-methylxanthine, whereas induction of ovulation by the two hormones is blocked by indomethacin. GnRH and LH differ, therefore, in the mechanism leading to PGE formation, but thereafter share a common mechanism responsible for oocyte maturation and independently for induction of ovulation.     

Allelic polymorphism of FMR1 gene CGG-repeat region in patients with impairment of natural and stimulated ovulation

The frequency of heterozygote carriers of (risk zone, alleles of FMR1 gene (40-47 CGG-repeats) was significantly higher in group of patients with ovarian dysfunction than in control group I. The tendency for higher frequency of those alleles was observed in patients with "poor response" to superovulation induction in IVF cycles. The average number of oocytes and follicles, which was obtained after stimulation of superovulation, was significantly decreased in FMR1 gene "risk zone" alleles carriers compared to patients with normal alleles of FMR1 gene. The average general dosage of exogenous gonadotrophin, necessary for superovulation induction was significantly higher in heterozygote carriers of FMR1 gene "risk zone" alleles than in patients with normal genotype. Thereby, the FMR1 gene "risk zone" alleles can be one of the hereditary susceptibility factors of impairment nature and stimulated ovulation.     

Veterinary clinical application of GnRH--questions of efficacy

The efficacy of GnRH treatments are reviewed in relation to prevention of embryo mortality, control of follicle development in synchronization programmes using PG as luteolysin, induction of ovulation in post-partum anoestrus and in bovine cystic ovarian disease. It is suggested that in cattle that GnRH is effective in increasing pregnancy rates when given either at the time of insemination (first or repeat) or between days 11 and 14 after insemination. Evidence is also presented for positive effects on pregnancy rates in sheep, mares and sows. Use of GnRH as an integral part of synchronising regimens where it is given 7 days before PG and then again 48-60 h after PG appears to be effective in increasing the synchrony of ovulation in controlled breeding programmes. The main synchronizing effect seems to reside in the second GnRH injection whereas the importance of the first is in prolonging the luteal phase in those cows treated late in the cycle. The published work on the potential use of GnRH to induce ovulation in anovulatory cattle is reviewed. Neither bolus dose injections, pulsatile, continuous infusion, nor controlled release formulations of GnRH, have yet proved effective in inducing fertile ovulations in a predictable or consistent manner. It is suggested that this is due to the variability of follicular status when treatment is initiated. GnRH is commonly used in the treatment of bovine cystic ovarian disease. However, although stimulating ovulation/luteinisation of a new follicle and luteinisation of the cyst, fertility of treated cattle remains very poor and it is suggested that a better understanding of the disease is needed before more effective treatments can be developed.     

Endocrine and ultrasound evaluation of the response to PGF 2alpha and GnRH given at different stages of the luteal phase in cyclic ewes

To investigate the effects of prostaglandin (PGF 2alpha) plus GnRH at different stages of the luteal phase 13 ewes received PGF 2alpha on Day 9 of the synchronized cycle, followed 36 h later by GnRH. This control regimen resulted in ovulation and normal corpus luteum (CL) function. In the next cycle, the ewes were treated simultaneously with PGF 2alpha and GnRH either on Day 4 (early, n = 7) or Day 9 (late, n = 6). Ovarian activity was monitored daily by ultrasonography, and blood samples were obtained to monitor hormonal patterns. Size of the largest follicle present when GnRH was administered was similar in all groups, but the preceding growth rate was greatest for the early group. In the 36 h after injection of PGF 2alpha, serum progesterone (P4) had declined to basal levels in the control cycles when GnRH was administered, but P4 concentrations were higher in the early group and were highest in the late group when the GnRH was administered with PGF 2alpha. The LH surges induced by GnRH were highest in the control cycles, and were lower in the 2 treated groups. In the early group, 6 of 7 ewes demonstrated ovulation within 48 h of GnRH, resulting in the formation of normal CL. In the late group, ovulation was delayed for about 5 d in 4 of 6 ewes, and subsequent luteal function was normal; no ovulation was detected in the other 2 ewes of this group, but the follicles became luteinized, resulting in a normal P4 profile in one and subnormal in the other. These results suggest that follicles present during the early luteal phase are capable of ovulating and forming fully functional CL in response to exogenous GnRH. In contrast, follicles present during the late luteal phase fail to ovulate in response to GnRH while P4 levels are high, even though the LH stimulus is adequate; however, these follicles persist and subsequently ovulate after P4 levels have decreased. Therefore, the endocrine milieu to which a follicle was exposed may be more important than its size in determining its ability to undergo ovulation and development into a normal CL.     

Associations between the manipulation of patterns of follicular development and fertility in cattle

The wave-like patterns of ovarian follicular development in cattle can be manipulated by shortening the luteal phase with prostaglandin F2alpha (PGF), lengthening the period of follicle dominance with progesterone or curtailing follicle development with GnRH or oestradiol as 17beta, benzoate or cypionate. These hormones can also be used to synchronise ovulation allowing timed inseminations without detected oestrus. Progesterone, PGF, GnRH and oestradiol benzoate have each been used to increase conception rates in some situations, but their use has reduced them in others. For example, inseminations made within 96 h of a single injection of PGF administered during the luteal phase were associated with increased conception rates in dairy cows whereas double injection protocols reduced conception rates. The three forms of oestradiol and GnRH have greater effects on follicular development following divergence and dominance than following wave emergence. This can mean that follicles of differing maturity will be present about 7 days later and can result in varied intervals to the onset of oestrus following a PGF injection. The consequent variation in ovulation time can be reduced by injecting GnRH or an oestradiol during pro-oestrus. This means that some less mature follicles will ovulate, forming corpus luteum (CL) associated with a slower rise in plasma progesterone and lower mid-luteal concentrations. The lower conception rates recorded with single timed inseminations with synchronised ovulations have been associated with increased prevalences of short cycles in lactating dairy cows (with GnRH), with long luteal phases in cows and heifers (with oestradiol benzoate) and with embryo loss following positive pregnancy diagnosis (as with Ovsynch in lactating Holstein cows). Extensive Canadian studies have demonstrated that these same hormones can be successfully used without these limitations and reliably obtaining conception rates over 50% and up to 70% in beef cattle that have been supplemented with a progestin during the period of ovarian follicle synchronisation. The inherently lower fertility of Holstein cows during early lactation may be contributing to the reduced effectiveness of hormonal treatments for synchronised follicle development and ovulation. The role of reduced dose rates of GnRH in compromising this effectiveness needs to be determined if the potential of these treatments realised with beef cattle is to be achieved with lactating Holstein cows.     

Prevention of multiple pregnancy during ovulation induction.

Gonadotrophin ovulation induction is currently used for a heterogeneous group of ovulation disorders and unexplained infertility. In the United States it is reported that multiple pregnancy rates of greater than 30% occur as a result of ovulation induction, most commonly after controlled ovarian hyperstimulation and intrauterine insemination. Treatment strategies to reduce the incidence of multiple pregnancies on ovulation induction programs can be targeted to reducing multiple follicular development and subsequent ovulation by a more aggressive cancellation policy, follicle reduction by fine needle aspiration or conversion to IVF; or dealing with the problem of multiple gestation after it has occurred (i.e., multifetal pregnancy reduction). The procedures and abilities exist to resolve this problem. What is needed are appropriate treatment guidelines and well constructed trials to demonstrate that higher order multiple pregnancies can be substantially reduced and/or eliminated without compromising a couple's chances to conceive.     

Participation of ezrin in bacterial uptake by trophoblast giant cells

A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.     

Pulsatile infusion of gonadotrophin releasing hormone (GnRH): investigative and therapeutic applications

Normal gonadotrophin secretion, and therefore normal ovarian function, depend on delivery to the pituitary of the hypothalamic neuropeptide gonadotrophin releasing hormone (GnRH) in a pulsatile pattern. In the mid-follicular phase of the menstrual cycle, for example, discrete pulses of luteinizing hormone (LH) can be observed at approximately 90 min intervals. Many disorders of ovulation are caused by abnormalities of this natural pulsed signal. We have developed and used a small portable infusion pump to deliver GnRH to women with hypothalamic amenorrhoea; our studies, and those of other groups, have shown that successful ovulation and pregnancy result from such treatment. The results of treatment at St Mary's Hospital show that 16 women with hypogonadotrophic amenorrhoea received a total of 31 cycles of treatment with pulsatile GnRH; 25 (81%) of these cycles were ovulatory and 11 of the 14 women who were trying to conceive became pregnant. There was only one multiple pregnancy (twins).     

Reproductive hormones associated with the ovarian cyst response to GnRH

Eighteen cows with ovarian cysts were administered 100 mug of GnRH and bled prior to treatment, at half hour intervals for 4 hours posttreatment and on days 1, 5 and 9 posttreatment. Blood plasma was analyzed for estradiol-17beta, progesterone and LH by radioimmunoassay. Response to treatment was recorded as positive if ovulation was detected within 30 days posttreatment. Fourteen cows (78%) initiated ovarian cycles by 30 days posttreatment. Mean pretreatment concentrations of estradiol-17beta, progesterone and LH and the GnRH induced LH release were not different for positive or no response cows. However, all seven cows that had pretreatment progesterone concentrations greater than 1.0 ng/ml had a positive response to treatment. Eight of the remaining eleven cows had a progesterone response (mean progesterone concentrations on days 5 and 9 posttreatment) greater than 1.0 ng/ml; seven had a positive response to treatment. In summary, most cows with ovarian cysts administered GnRH will initiate ovarian cycles within 30 days if: 1) pretreatment progesterone concentrations are greater than 1.0 ng/ml or 2) if progesterone response is greater than 1.0 ng/ml.     

Allelic polymorphism of the CGG repeat region in the <Emphasis Type="Italic">FMR1</Emphasis> gene in patients with impaired natural and stimulated ovulation

The frequency of heterozygote carriers of risk zone alleles of the FMR1 gene (40–47 CGG repeats) was significantly higher in the group of patients with ovarian dysfunctions compared to control group I. The frequency of these alleles shows an increasing tendency in patients poorly responding to superovulation induction in IVF cycles. The average number of oocytes and follicles obtained from the stimulation of superovulation was significantly decreased in FMR1 gene heterozygous risk zone allele carriers as compared to patients with normal alleles of the FMR1 gene. The general average dosage of exogenous gonadotrophin necessary for superovulation induction was significantly higher in heterozygote carriers of FMR1 gene risk zone alleles than in patients with normal genotype. As well, the FMR1 gene risk zone alleles can be one of the hereditary susceptibility factors of impaired natural and stimulated ovulation.     

Controlled ovarian stimulation and ultrasound guided follicular aspiration in the baboon (Papio cynocephalus anubis)

The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and highly purified human FSH (hphFSH) with follicular development and oocyte maturation. An ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33-34 days). A long-acting GnRHa implant containing goserelin acetate was placed s.c. on days 22-24 of their menstrual cycle. Daily hphFSH (75 IU im) treatments were started approximately 10 days following menses. When the majority of the follicles were > or = 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of the oocytes and ovulation. 30 to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected (average: 17). 91% of the oocytes were morphologically normal indicating that they were appropriate for in vitro insemination.     

The macaque model for in vitro fertilization: superovulation techniques and ultrasound-guided follicular aspiration

Prior methods for macaque in vitro fertilization (IVF) have incorporated laparoscopy and/or laparotomy as the primary means for oocyte recovery. Sonographic techniques, as used with human IVF, have been applied to the macaque, both for monitoring the response to hyperstimulation and for follicular aspiration prior to ovulation. Pergonal (hMG) was administered for 7 or 8 days beginning on cycle day 1 or 2 or for 6 days beginning on cycle day 3. This was followed by Pregnyl (hCG) prior to follicular aspiration. The quality of oocytes recovered from the 6-day treatment group was considerably better than those treated for greater than or equal to 7 days. It was concluded that ultrasound can provide a reliable means for documenting the response to ovarian stimulation and the successful transabdominal aspiration of multiple follicles.     

Aromatase inhibitors in post-menopausal endometriosis

Background

Serum anti-Mullerian hormone (AMH) is currently considered the best marker of ovarian reserve and of ovarian responsiveness to gonadotropins in in-vitro fertilization (IVF). AMH assay, however, is not available in all IVF Units and is quite expensive, a reason that limits its use in developing countries. The aim of this study is to assess whether the "ovarian sensitivity index" precisely reflects AMH so that this index may be used as a surrogate for AMH in prediction of ovarian response during an IVF cycle.

Methods

AMH serum levels were measured in 61 patients undergoing IVF with a "long" stimulation protocol including the GnRH agonist buserelin and recombinant follicle-stimulating hormone (rFSH). Patients were divided into four subgroups according to the percentile of serum AMH and their ovarian stimulation was prospectively followed. Ovarian sensitivity index (OSI) was calculated dividing the total administered FSH dose by the number of retrieved oocytes.

Results

AMH and OSI show a highly significant negative correlation (r = -0.67; p = 0.0001) that is stronger than the one between AMH and the total number of retrieved oocytes and than the one between AMH and the total FSH dose.

Conclusions

OSI reflects quite satisfactory the AMH level and may be proposed as a surrogate of AMH assay in predicting ovarian responsiveness to FSH in IVF. Being very easy to calculate and costless, its use could be proposed where AMH measurement is not available or in developing countries where limiting costs is of primary importance.     

Induction of luteal regression in the marmoset monkey (Callithrix jacchus) by a gonadotrophin-releasing hormone antagonist and the effects on subsequent follicular development

Doses of 100 or 200 micrograms of a novel GnRH antagonist ([N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8-D-Ala10]NH2 GnRH) (4 animals/dose) were administered on Days 10/11 of the luteal phase and induced a marked suppression of circulating bioactive LH and progesterone concentrations within 1 day of treatment (P less than 0.01). Thereafter, progesterone concentrations remained low or undetectable until after the next ovulation. Similar results were obtained when 200 micrograms antagonist were given on Days 5/6 of the luteal phase (N = 4). The interval from injection of antagonist (200 micrograms but not 100 micrograms) to ovulation (based on a rise in progesterone above 10 ng/ml) was significantly longer than that from prostaglandin-induced luteal regression to ovulation in control cycles (N = 4/treatment) (range, 13-15 days after antagonist vs 8-10 days after prostaglandin, P less than 0.01). This delay of 4-5 days was equivalent to the duration for which LH concentrations were significantly suppressed by 200 micrograms antagonist when administered to ovariectomized animals (N = 3). Corpus luteum function during the cycle after GnRH antagonist treatment appeared normal according to the pattern of circulating progesterone. These results show that corpus luteum function and preovulatory follicular development in the marmoset monkey are dependent on pituitary gonadotrophin secretion.     

Milder is better? advantages and disadvantages of "mild" ovarian stimulation for human in vitro fertilization

In the last decades, several steps have been made aiming at rendering human IVF more successful on one side, more tolerable on the other side. The "mild" ovarian stimulation approach, in which a lower-than-average dose of exogenous gonadotropins is given and gonadotropin treatment is started from day 2 to 7 of the cycle, represents a significant step toward a more patient's friendly IVF. However, a clear view of its virtues and defects is still lacking, because only a few prospective randomized trials comparing "mild" vs. conventional stimulation exist, and they do not consider some important aspects, such as, e.g., thawing cycles. This review gives a complete panorama of the "mild" stimulation philosophy, showing its advantages vs. conventional ovarian stimulation, but also discussing its disadvantages. Both patients with a normal ovarian responsiveness to exogenous gonadotropins and women with a poor ovarian reserve are considered. Overall, we conclude that the level of evidence supporting the use of "mild" stimulation protocols is still rather poor, and further, properly powered prospective studies about "mild" treatment regimens are required.     

Extra-pituitary action of gonadotropin releasing hormone: possible application

Chronic administration of a potent gonadotropin releasing hormone inhibits ovulation in women. The suppression of gonadal function during long term treatment with the GnRH analogues is ascribable to inhibition of gonadotropin secretion caused by the down regulatory action of the decapeptide at the pituitary level. Reduced progesterone production with premature onset of menstruation has been observed in women injected with the agonist during the midluteal phase. The decapeptide however, has no effect onin vitro human ovarian steroidogenesis. Specific receptors for GnRH have been located on rodent ovarian cells, but corpora lutea of rhesus monkey and human ovaries seem to lack these receptors. The luteolytic effect in women thus appears to be central in origin and not a direct effect on the corpus luteum. Recently, a superactive agonist of GnRH given around the peri-implantation period has been shown to terminate pregnancy in baboons. Monoclonal antibodies against GnRH administered during the same period in a fertile cycle also abrogated pregnancy in these animals. Using immuno-enzymatic techniques GnRH has been localized on the placenta. GnRH also exerts a stimulatory effect on hCG production by the placental villi maintained in culture. Addition of anti-luteinizing hormone releasing hormone antibodies blocks this effect completely. It seems that placenta is the only other tissue besides the pituitary where GnRH has probably a regulatory role in the human female.