Ghanim Almahbobi

Functional integrity of granulosa cells from polycystic ovaries

OBJECTIVE The polycystic ovarian syndrome is frequently associated with human infertility and is a partially characterized syndrome of unknown aetiology. The aim of this study was to describe the functional integrity of granulosa cells from polycystic ovaries.

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.

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.

OPTIMISING VITRIFICATION OF HUMAN OOCYTES USING MULTIPLE CRYOPROTECTANTS AND MORPHOLOGICAL AND FUNCTIONAL ASSESSMENT

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

Hyperexpression of epidermal growth factor receptors in granulosa cells from women with polycystic ovary syndrome

OBJECTIVE To compare the localization and quantitation of epidermal growth factor (EGF) receptor in granulosa cells from women with normal and polycystic ovaries. DESIGN Controlled, comparative study. SETTING Academic research laboratory. PATIENT(S) Forty-two women with normal or polycystic ovaries who attended our facility for the recovery of their immature or mature oocytes or for therapeutic purposes. INTERVENTION(S) Patients underwent intravaginal ultrasound-guided oocyte retrieval or laparoscopic follicular aspiration with or without prior stimulation. MAIN OUTCOME MEASURE(S) Quantitation of EGF receptor in granulosa cells. RESULT(S) Granulosa cells from polycystic ovaries expressed significantly higher levels of EGF receptor than granulosa cells from normal ovaries. In contrast with patients who were treated with clomiphene citrate, those who were treated with gonadotropins showed low levels of the receptor. However, the levels of the receptor in granulosa cells were not correlated with circulating levels of LH, FSH, progesterone, or E2. Immunolabeling of EGF receptor was confined to the cell membrane of granulosa cells. This receptor was fully functional, mediating the ligand-induced inhibition of E2 production in culture. CONCLUSION(S) These results provide further evidence supporting a possible role of EGF/transforming growth factor-alpha in the aberration of ovarian function in polycystic ovary syndrome.

Computer-based detection of neonatal changes to branching morphogenesis reveals different mechanisms of and predicts prostate enlargement in mice haplo- insufficient for bone morphogenetic protein 4

Early changes to branching morphogenesis of the prostate are believed to lead to enlargement of the gland in adult life. However, it has not been possible to demonstrate directly that alterations to branching during the developmental period have a permanent effect on adult prostate size. In order to examine branching morphogenesis in a quantitative manner in neonatal mice, a combination of imaging and computational technology was used to detect and quantify branching using bone morphogenetic protein 4 haplo‐insufficient mice that develop enlarged prostate glands in adulthood. Accurate estimates were made of six parameters of branching, including prostate ductal length and volume and number of main ducts, branches, branch points, and tips. The results show that the prostate is significantly larger on day 3, well before the emergence of the phenotype in older animals. The ventral prostate is enlarged because the number of main epithelial ducts is increased; enlargement of the anterior prostate in mutant animals occurs because there are more branches. These lobe‐specific mechanisms underlying prostate enlargement indicate the complex nature of gland pathology in mice, rather than a simple increase in weight or volume. This method provides a powerful means to investigate the aetiology of prostate disease in animal models prior to emergence of a phenotype in later life. Copyright

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.

A direct action for GH in improvement of oocyte quality in poor-responder patients

Declining female fecundity at later age and the increasing tendency for women to delay childbirth have lead to a drastic rise in the number of women seeking assisted reproductive technology. Many women fail to respond adequately to standard ovarian stimulation regimens, raising a significant therapeutic challenge. Recently, we have demonstrated that the administration of GH, as an adjunct to ovarian stimulation, has improved the clinical outcomes by enhancing the oocyte quality. However, the mechanism(s) by which GH facilitated this improvement is yet to be understood. This study aimed to determine these potential mechanism(s) through the use of immunofluorescent localisation of GH receptors (GHRs) on the human oocyte and unbiased computer-based quantification to assess and compare oocyte quality between women of varying ages, with or without GH treatment. This study demonstrates for the first time, the presence of GHRs on the human oocyte. The oocytes retrieved from older women showed significant decrease in the expression of GHRs and amount of functional mitochondria when compared with those from younger patients. More interestingly, when older patients were treated with GH, a significant increase in functional mitochondria was observed in their oocytes. We conclude that GH exerts a direct mode of action, enabling the improvement of oocyte quality observed in our previous study, via the upregulation of its own receptors and enhancement of mitochondrial activity. This result, together with recent observations, provides scientific evidence in support of the use of GH supplementation for the clinical management of poor ovarian response.

Changes in Ovarian Follistatin Levels During the Oestrous Cycle in Sheep may Serve as an Intraovarian Regulator

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day -1 and 0) to mid-luteal phase (days 11-13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.

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.