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.

Mid-luteal serum progesterone concentrations govern implantation rates for cryopreserved embryo transfers conducted under hormone replacement.

This study explores the relevance of mid-luteal serum hormonal concentrations in cryopreserved embryo transfer cycles conducted under hormone replacement therapy (HRT) control and which involved single-embryo transfer (SET) of 529 vitrified blastocysts. Widely ranging mid-luteal oestradiol and progesterone concentrations ensued from the unique HRT regimen. Oestradiol had no influence on clinical pregnancy or live birth rates, but an optimal progesterone range between 70 and 99 nmol/l (P < 0.005) was identified in this study. Concentrations of progesterone below 50 nmol/l and above 99 nmol/l were associated with decreased implantation rates. There was no clear interaction between oestradiol and progesterone concentrations but embryo quality grading did show a significant influence on outcomes (P < 0.001 and P = 0.002 for clinical pregnancy and live birth rates, respectively). Multiple comparison analysis showed that the progesterone effect was influential regardless of embryo grading, body mass index or the woman's age, either at vitrification or at cryopreserved embryo transfer. The results support the argument that careful monitoring of serum progesterone concentrations in HRT-cryopreserved embryo transfer is warranted and that further studies should explore pessary adjustments to optimize concentrations for individual women to enhance implantation rates.

Follicle recruitment determines IVF productivity rate via the number of embryos frozen and subsequent transfers.

IVF productivity rate is an index defined as the sum of all live births from either fresh or frozen embryo transfers arising from a single oocyte collection. This retrospective analysis over 9 continuous years used this index to understand the potential impact on pregnancy rates of milder stimulation regimens with associated reduced egg numbers. The productivity rate per collection increased in a linear and significant rate as more oocytes were recovered, more embryos frozen and more frozen embryo transfers contributed to pregnancy. This observation was true for women aged <35 years and less so for women aged 35-39 years but not for women aged 40 years and older. The contribution of frozen embryo transfer to the productivity rate rose in a linear manner, reaching over 40% of all live births with nine oocytes. The number of live births per oocyte, pronuclear embryos and thawed embryos decreased significantly but the number of live births per embryo transferred (fresh or frozen) rose with rising oocyte numbers, reflecting increasing opportunity for embryo selection. This study suggests that optimal benefits with minimal risks are gained from a model that includes both fresh and frozen transfers under stimulation generating between 8 and 12 eggs.

Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates.

Occasionally, clinical scenarios arise where embryos, previously cryostored and warmed, need to be recryopreserved. The outcome of 30 such transfer cycles from 25 women where embryos were recryopreserved is detailed. In 16 cases, embryos were initially cryopreserved by slow freezing and in 14 cases by vitrification. The cryopreservation stages were the pronuclear stage (n = 16), day-3 cleavage stage (n = 12), blastocyst (n = 1) and oocytes (n = 1). All recryopreservation was by Cryotop-based vitrification. From this mixed source, 30/31 twice-cryopreserved embryos survived warming and were transferred, resulting in 13 pregnancies, 11 deliveries with normal gestational age and birthweight, one pre-term birth at 33 weeks and two miscarriages. There were no malformations reported for the live births. Recryopreservation using vitrification by CryoTop has been used in a variety of clinical scenarios to preserve surplus cryopreserved embryos. The current study, although limited in numbers, resulted in high survival rates, clinical pregnancy rates similar to once-cryopreserved embryos and healthy live births independently of the initial stage and cryopreservation method. The technique may increasingly be applicable to elective single-embryo transfer and blastocyst transfer to maximize the pregnancy rate while minimizing the number of cryopreserved embryo transfers.

Targeted gonadotrophin stimulation using the PIVET algorithm markedly reduces the risk of OHSS.

PIVET Medical Centre has developed an empirical algorithm for the dose of FSH administration based upon day-2 FSH, antral follicle count, anti-Müllerian hormone, body mass index, age and smoking parameters in an attempt to reduce the incidence of ovarian hyperstimulation syndrome particularly in at-risk women with elevated antral follicle count and anti-Müllerian hormone. The algorithm utilized the incremental dosage capabilities of the recombinant FSH pens to fine-tune the daily concentration of FSH. Application of the algorithm aimed to minimize any form of excessive follicle recruitment that necessitated increased clinical awareness. The measure used to assess the impact of the algorithm was the number of women who, after oocyte retrieval, were considered to be potentially at risk of any degree of OHSS and were allocated to increased monitoring. Compared with the previous 20-month period, introduction of the algorithm significantly reduced both the incidence of referral for increased monitoring, treatment for OHSS and the incidence of freeze-all cycles (all P < 0.05). This was particularly focused on those considered to be at risk without reducing the fresh cycle pregnancy rate.

Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation.

One concern during intracytoplasmic sperm injection (ICSI) is that selected spermatozoa may have increased levels of DNA damage; however, the available testing for this is largely destructive in nature and therefore unsuitable as a tool for sperm selection. One alternative selection process that has previously achieved pregnancies is the hypo-osmotic swelling test (HOST). This study reports that low HOST values of neat semen samples were significantly (P<0.001) associated with increased DNA damage identified by the DNA fragmentation index (DFI) from the sperm chromatin structure assay as well as the TdT-mediated dUTP nick-end labelling (TUNEL) assay. The HOST value was highly predictive of an abnormal DFI value by receiver operating characteristic curve analysis (P<0.001). Furthermore, when individual spermatozoa were assessed for both HOST status and DNA fragmentation by TUNEL, the key HOST-induced tail-swelling grades D, E and F were most commonly associated with high HOST values and were significantly (P<0.001) associated with minimal DNA damage regardless of the DNA status of the ejaculate. The application of HOST may be a valuable tool in the routine identification and selection of viable, DNA-intact individual spermatozoa for ICSI after further research to demonstrate its efficacy and safety.

Growth hormone supplementation improves implantation and pregnancy productivity rates for poor-prognosis patients undertaking IVF.

In a sequential crossover study of IVF conducted from 2002 to 2006, growth hormone (GH) supplementation was assessed in poor-prognosis patients, categorized on the basis of past failure to conceive (mean 3.05 cycles) due to low response to high-dose stimulation (<3 metaphase II oocytes) or poor-quality embryos. Pregnancy rates in both fresh and frozen transfer cycles and the total productivity rates (fresh and frozen pregnancies per egg collection) were compared. In all, 159 patients had 488 treatment cycles: 221 with GH and 241 without GH. These cycles were also compared with 1572 uncategorized cycles from the same period. GH co-treatment significantly improved the clinical pregnancy rate per fresh transfer (P<0.001) as well as per frozen-thawed embryo derived from GH cycles (P<0.05) creating a highly significant productivity rate (P<0.001). The effect was significant across all age groups, especially in younger patients, and was independent of stimulation modality or number of transfers. GH cycles resulted in significantly more babies delivered per transfer than non-GH cycles (20% versus 7%; P<0.001) although less than the uncategorized cycles (53%). The data uniquely show that the effect of GH is directed at oocyte and subsequent embryo quality.