The Evolving Concept of Poor-Prognosis for Women Undertaking IVF and the Notion of Growth Hormone as an Adjuvant; A Single-Center Viewpoint.

IVF is currently regarded as a successful new technology with the number of IVF children currently well over 8 million worldwide. This has been achieved by an explosive plethora of facilities. However, from its earliest history, IVF has been beset by poor-prognosis on a treatment cycle basis, an aspect which has been a constant feature for the majority of treatments to this stage. The 2019 Australian and New Zealand Assisted Reproduction Database (ANZARD) report shows that IVF clinics have live birth productivity rates (from combined initiated fresh and frozen cycles) ranging from 9.3 to 33.2%. Over the past 40 years there have been a number of innovations which have steadily moved the success rates forward, but progress is held back by an intransigent group of women who can be classified as being poor-prognosis from one or more adverse factors, namely advanced age (>40 years), poor ovarian response (POR) to ovarian stimulation, inability to generate high quality blastocyst-stage embryos, recurrent implantation failure, or recurrent early pregnancy losses. A number of strategies are variously applied including the use of recombinant growth hormone (GH) adjuvant therapy. Our retrospective studies at PIVET over the past decade show a 6.2-fold chance of live birth for fresh cycle embryo transfers following GH injections of 1-1.5 IU daily given for 3-6 weeks in the lead-up to the trigger for ovum pick-up. We have also recently reported the live birth rates from frozen embryo transfers utilizing those blastocyst embryos generated under GH influence and showed the live birth rate was 2.7-fold higher in a carefully matched poor-prognosis group. This experience has been compared to the total 42 GH studies reported since the year 2000, the majority matching those of PIVET with significant increases in both oocyte and embryo utilization rates but only ~50% are followed by elevated live birth rates. We argue that this discrepancy relates to failure in addressing other causes of poor-prognosis along with the wastage of transferring more than a single embryo in the fresh cycle, when ANZARD data indicates a significantly higher chance of live birth from frozen embryo transfers.

The Concept of Growth Hormone Deficiency Affecting Clinical Prognosis in IVF.

The current understanding of human growth hormone (hGH; here GH) action is that the molecule is a 191-amino acid, single-chain polypeptide that is synthesized, stored and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. It can be classified as a protein (comprising more than 50 amino acids) but true proteins have tertiary and quaternary chains creating a more complex structure, hence GH is usually classified as a polypeptide. GH is normally secreted at night during sleep and promotes skeletal, visceral and general body growth through the action of somatomedins or IGFs, notably IGF-1. In some tissues, GH action is directed via specific receptors GHRs; these are most abundant in liver, adipose and muscle tissues but have also been shown in granulosa cells, testicular tissues and on the oocyte, as well as in glandular cells of the luteal phase endometrium and decidua; such findings being recent and minimally researched to now. Following engagement with its receptor, the transduction process activates multiple signaling proteins. These all lead to extensive metabolic and mitogenic (growth promoting) responses. Clinically, GH is known to have an important role in pubertal development and is a key hormone for the vigor associated with adolescence and early adult life stages but has a faded presence and role for later adulthood, beyond age 30 years, and is minimally detected in advanced age, beyond 40 years. In association with the rapidly increasing trend for delaying reproduction beyond age 35 years, GH is being widely researched now as a potential adjuvant for infertility treatment in this group who, studies consistently show, have a poorer prognosis than younger females when relying on autologous oocytes. The idea that the age-related reduction in fertility prognosis is a feature of growth hormone deficiency is supported by our studies showing an elevated binding protein IGFBP-3/IGF-1 ratio and this can be reduced to a normal range (matching younger, good prognosis women) by the administration of GH as an adjuvant.

Growth hormone during in vitro fertilization in older women modulates the density of receptors in granulosa cells, with improved pregnancy outcomes.

OBJECTIVE: To study the effect of aging and granulosa cell growth hormone receptor (GHR) expression, and the effect of growth hormone (GH) co-treatment during IVF on receptor expression. DESIGN: Laboratory study. SETTING: University. PATIENT(S): A total of 445 follicles were collected from 62 women undergoing standard infertility treatment. INTERVENTION(S): Preovulatory ovarian follicle biopsies of granulosa cells and follicular fluid. MAIN OUTCOME MEASURE(S): Older women with a poor ovarian reserve were co-treated with GH to determine the effect of the adjuvant during IVF on the granulosal expression density of FSH receptor (FSHR), LH receptor (LHR), bone morphogenetic hormone receptor (BMPR1B), and GHR. Ovarian reserve, granulosa cell receptor density, oocyte quality, and pregnancy and live birth rates were determined. RESULT(S): Growth hormone co-treatment increased the receptor density for granulosal FSHR, BMPR1B, LHR, and GHR compared with the non-GH-treated patients of the same age and ovarian reserve. Growth hormone co-treatment increased GHR density, which may increase GHR activity. The GH co-treatment was associated with a significant increase in pregnancy rate. CONCLUSION(S): Growth hormone co-treatment restored the preovulatory down-regulation of FSHR, BMPR1B, and LHR density of the largest follicles, which may improve the maturation process of luteinization in older patients with reduced ovarian reserve. The fertility of the GH-treated patients improved.

Granulosa Cell Apoptosis in the Ovarian Follicle-A Changing View.

Recent studies challenge the previous view that apoptosis within the granulosa cells of the maturing ovarian follicle is a reflection of aging and consequently a marker for poor quality of the contained oocyte. On the contrary, apoptosis within the granulosa cells is an integral part of normal development and has limited predictive capability regarding oocyte quality or the ensuing pregnancy rate in in vitro fertilization programs. This review article covers our revised understanding of the process of apoptosis within the ovarian follicle, its three phenotypes, the major signaling pathways underlying apoptosis as well as the associated mitochondrial pathways.

Involvement of Bone Morphogenetic Proteins (BMP) in the Regulation of Ovarian Function.

Primordial germ cells migrate to the fetal gonads and proliferate during gestation to generate a fixed complement of primordial follicles, the so-called ovarian reserve. Primordial follicles comprise an oocyte arrested at the diplotene stage of meiosis, surrounded by a layer of pregranulosa cells. Activation of primordial follicles to grow beyond this arrested stage is of particular interest because, once activated, they are subjected to regulatory mechanisms involved in growth, selection, maturation, and ultimately, ovulation or atresia. The vast majority of follicles succumb to atresia and are permanently lost from the quiescent or growing pool of follicles. The bone morphogenetic proteins (BMPs), together with other intraovarian growth factors, are intimately involved in regulation of follicle recruitment, dominant follicle selection, ovulation, and atresia. Activation of primordial follicles appears to be a continuous process, and the number of small antral follicles at the beginning of the menstrual cycle provides an indirect indication of ovarian reserve. Continued antral follicle development during the follicular phase of the menstrual cycle is driven by follicle stimulating hormone (FSH) and luteinizing hormone (LH) in conjunction with many intraovarian growth factors and inhibitors interrelated in a complex web of regulatory balance. The BMP signaling system has a major intraovarian role in many species, including the human, in the generation of transcription factors that influence proliferation, steroidogenesis, cell differentiation, and maturation prior to ovulation, as well as formation of corpora lutea after ovulation. At the anterior pituitary level, BMPs also contribute to the regulation of gonadotrophin production.

The effect of ovarian reserve and receptor signalling on granulosa cell apoptosis during human follicle development.

The poor oocyte quality in older women has previously been linked to the depletion of the ovarian reserve of primordial follicles and an increase in granulosal apoptosis. Granulosa cells were collected from 198 follicles and individually analysed by flow cytometry. In the young IVF patients, the level of apoptosis was inversely proportional to the expression of bone morphogenetic protein (BMPR1B) and follicle stimulating hormone (FSH) receptors. Conversely, in the older patients this relationship became dysregulated. In the older patients, at the time of preovulatory maturation, the reduced apoptosis reflects the poor mitogenic growth turnover rate of healthy follicles rather than the death rate in an atretic follicle. Restoring an optimum receptor density and down-regulation of receptors may improve oocyte quality and the pregnancy rate in older women.

Infertility and ovarian follicle reserve depletion are associated with dysregulation of the FSH and LH receptor density in human antral follicles.

The low take-home baby rate in older women in Australia (5.8%) undergoing IVF (5.8%) is linked to the depletion of the ovarian reserve of primordial follicles. Oocyte depletion causes an irreversible change to ovarian function. We found that the young patient FSH receptor and LH receptor expression profile on the granulosa cells collected from different size follicles were similar to the expression profile reported in natural cycles in women and sheep. This was reversed in the older patients with poor ovarian reserve. The strong correlation of BMPR1B and FSH receptor density in the young was not present in the older women; whereas, the LH receptor and BMPR1B correlation was weak in the young but was strongly correlated in the older women. The reduced fertilisation and pregnancy rate was associated with a lower LH receptor density and a lack of essential down-regulation of the FSH and LH receptor. The mechanism regulating FSH and LH receptor expression appears to function independently, in vivo, from the dose of FSH gonadotrophin, rather than in response to it. Restoring an optimum receptor density may improve oocyte quality and the pregnancy rate in older women.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility.

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry. A decline in BMPR1B density occurred at the time of dominant follicle selection and during the terminal stage of folliculogenesis in the 23-30 y good ovarian reserve patients. The 40+ y poor ovarian reserve patients experienced a reversal of this pattern. The results demonstrate an association between age-induced depletion of the ovarian reserve and BMPR1B receptor density at the two critical time points of dominant follicle selection and pre-ovulatory follicle maturation. Dysregulation of BMP receptor signalling may inhibit the normal steroidogenic differentiation required for maturation in older patients.

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep.

The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations.