The Role of One-Carbon Metabolism and Methyl Donors in Medically Assisted Reproduction: A Narrative Review of the Literature.

One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.

The impact of cryopreservation on both sperm HPV-negative and positive subtypes.

It is well known that various human papillomavirus (HPV) genotypes are present in semen specimens. Also, it has been demonstrated that sperm parameters are negatively affected when HPV infection is present in the sperm sample. Besides all these, the effect of cryopreservation on HPV sensitivity and resistance is not known. The aim of the present study is to evaluate first the prevalence of HPV and secondly to elucidate whether cryopreservation of sperm HPV-positive samples has any effect on the viability of HPV. For this purpose, a cohort of 78 sperm specimens was used from a respective number of patients. After giving informed consent, semen analysis was performed. Each sperm sample was divided into four equal aliquots. The first one (fresh) was evaluated for the prevalence of HPV, while the other three aliquots were cryopreserved by adding an equal quantity of cryoprotectant and plunged into the LN. Each of the three aliquots was thawed 3, 6, and 12 months later, respectively, so as to evaluate whether there is a time-resistance period of HPV prevalence. HPV infection was found to be in eleven sperm samples, demonstrating a 14.1% (11/78) HPV prevalence. Among the HPV-positive samples, six of them were high-risk and the remaining were low-risk genotypes. Moreover, the high-risk fresh samples demonstrated higher motility values than the low-risk samples (60% ± 2.7 vs 45.6% ± 3.7, p < .05), while semen volume in the high-risk samples was significantly lower than the respective volume in the low-risk samples (2.26 ± 0.2ml vs 3.5 ± 0.6ml, p < .05). Interestingly, cryopreservation of the HPV-positive samples resulted in the sustainability and time-resistance of HPV in all high-risk HPV-positive samples, something that was not the case with the low-risk HPV-positive samples. Conclusively, sperm samples infected with high-risk HPV, demonstrate lower sperm parameters and time-resistance activity during cryopreservation.

The Impact of SARS-CoV-2 on Sperm Cryostorage, Theoretical or Real Risk?

Cryopreservation of human gametes and embryos as well as human reproductive tissues has been characterized as an essential process and aspect of assisted reproductive technology (ART). Notably, sperm cryopreservation is a fundamental aspect of cryopreservation in oncological patients or patients undergoing gonadotoxic treatment. Given that there is a risk of contamination or cross-contamination, either theoretical or real, during the procedures of cryopreservation and cryostorage, both the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) have provided updated guidelines for preventing or reducing the contamination risk of sexually transmitted viruses. Given the ongoing and worldwide COVID-19 pandemic, there is considerable interest in what measures should be taken to mitigate SARS-CoV-2 contamination during cryopreservation and cryostorage of semen samples. The SARS-CoV-2 virus is the virus that causes COVID-19, and whose transmission and infection is mainly aerosol-mediated. Several ART professional societies, including ESHRE and ASRM have proposed measures to mitigate the spread of the SARS-CoV-2 virus. Whether the proposed safety directives are enough to mitigate the possible SARS-CoV-2-contamination of sperm samples during cryopreservation or whether the policies should be re-evaluated will be discussed in this review. Additionally, insights regarding the possible impact of COVID-19 vaccination on the safety of sperm cryopreservation will be discussed.

Effect of adiponectin on estradiol and progesterone secretion from human luteinized granulosa cells in vitro.

Ιnformation on the role of adiponectin in human ovarian steroidogenesis is limited. The present study aimed to investigate the effect of different doses of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells in culture. Granulosa cells, obtained from women undergoing in vitro fertilization (IVF) treatment, were pre-incubated for 24 h and then cultured for 48 h. Adiponectin was used in 3 doses, i.e., 5, 10, and 100 µg/ml alone and in combinations with FSH (10 and 100 ng/ml). Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. Adiponectin after 48 h of culture stimulated the secretion of estradiol and, to a lesser extent, progesterone in a dose-dependent manner. FSH showed a variable effect on steroidogenesis. However, when the low dose FSH was combined with adiponectin, estradiol, and progesterone secretion were increased disproportionally to the dose of adiponectin. With the high dose FSH, the positive effect of adiponectin on FSH-induced estradiol secretion was less pronounced, while the effect on progesterone secretion was negligible. This study shows for the first time a stimulatory effect of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is suggested that adiponectin plays a paracrine role in human ovarian steroidogenesis by sensitizing the granulosa cells to FSH.

Exogenous progesterone for LH surge prevention is redundant in ovarian stimulation protocols.

During ovarian stimulation for IVF-embryo transfer treatment, a premature LH surge may lead to progesterone elevation that disrupts endometrial maturation and affects the probability of pregnancy following fresh embryo transfer. Preventing this LH surge and progesterone elevation using gonadotrophin-releasing hormone (GnRH) analogues is considered a standard practice. The same policy applies to cycles in which the 'freeze-all' protocol has been selected from the outset (e.g. donors), but the need for this has not been discussed. Moreover, in 'freeze-all' cycles, exogenous progesterone administration tends to replace GnRH antagonists, without reducing efficacy after embryo transfer in frozen-thawed cycles. Nevertheless, as exogenous progesterone is expected to have the same impact on the endometrium as endogenous progesterone, it is clear that, unlike in fresh cycles, in 'freeze-all' cycles an endogenous LH surge prevention does not seem necessary. Therefore, both GnRH antagonists and exogenous progesterone appear to be redundant in 'freeze-all' cycles, and in this context the indications for the use of GnRH analogues in ovarian stimulation protocols need to be revisited.

The effect of a GnRH antagonist on follicle maturation in normal women.

RESEARCH QUESTION: Ganirelix is a gonadotrophin-releasing hormone (GnRH) antagonist used for the prevention of premature LH surge during ovarian stimulation. What is the impact of ganirelix on follicle maturation in normal women? DESIGN: Ten normally cycling women were investigated during two menstrual cycles, i.e. cycle 1 (control) and cycle 2 (ganirelix). During both cycles, daily blood samples were taken from day 2, while transvaginal ultrasound scans were performed on cycle days 8 and 10 and daily thereafter. During cycle 2, all women were given 0.25 mg/day subcutaneous injections of the GnRH antagonist ganirelix from day 2 until the day of the endogenous LH surge onset in cycle 1. RESULTS: During treatment with ganirelix, serum FSH and oestradiol concentrations remained stable, while those of LH decreased significantly on days 3, 4, 7 and 9 (P < 0.05) compared with controls. Nevertheless, there was no significant within-cycle variation in LH concentrations. From day 10 onwards, no follicle maturation was observed in cycle 2, in contrast to cycle 1. Ovulation occurred in 9 of 10 women in cycle 1. In cycle 2, ovulation was delayed by at least 1 week in eight women. Follicle growth and ovulation occurred in only one woman while on ganirelix treatment. CONCLUSIONS: This study demonstrates for the first time that in normal women dominant follicle selection failed during treatment with ganirelix. As there was a similar gonadotrophin profile in the two cycles, it is suggested that ganirelix interferes with the process of follicle selection by acting in the ovary.

Effect of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture.

Information on the role of resistin on steroidogenesis is limited to animal studies. The aim of this study was to investigate the effect of various doses of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. Granulosa cells were obtained from follicular fluid aspirated from 50 women undergoing in vitro fertilization (IVF) treatment. The cells were cultured for 48 h after a 24 h pre-incubation period. The effect of resistin at dosages 1, 10 and 100 ng/ml alone or in combinations with FSH (10 and 100 ng/ml) on steroidogenesis was investigated. Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. FSH treatment increased both estradiol and progesterone secretion. Resistin suppressed basal estradiol (at 1 ng/ml) and progesterone secretion (at all concentrations tested). When resistin (all concentrations) was combined with FSH (100 ng/ml), it eliminated the stimulatory effect of FSH on the secretion of estradiol and progesterone. This study indicates an inhibitory effect of resistin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is likely that this adipokine locally affects ovarian function in women. Abbreviations: 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; CAP1: cyclase-associated protein 1; DCN: decorin; FIZZ: Found in Inflammatory Zones; hCG: human chorionic gonadotropin; IGF1: insulin-like growth factor type 1; IVF: in vitro fertilization; PCOS: polycystic ovary syndrome; RIA: radioimmunoassay; ROR1: receptor tyrosine kinase-like orphan receptor-1; TLR4: Toll-like receptor 4.

Artificial oocyte activation: physiological, pathophysiological and ethical aspects.

Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca2+) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca+2 oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca+2 oscillations derive from a periodic release of Ca+2 from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca+2 oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure. Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca+2: Calcium; CAMKII: Ca+2/calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP3R: inositol-trisphosphate receptor; IP3: inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP2: phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr+2: strontium; TFF: total fertilization failure.

The Effect of Metformin on the Endometrium of Women with Polycystic Ovary Syndrome.

OBJECTIVES: To investigate the effect of metformin on endometrial receptivity in women with polycystic ovary syndrome (PCOS). METHODS: Twenty volunteer women with polycystic ovaries and oligomenorrhea were prospectively investigated. All women were treated with exogenous estradiol and progesterone to simulate a normal menstrual cycle (28-day duration) after GnRH-induced pituitary desensitization. Ten of the women received no other medication (group A, control), while the remaining 10 received metformin (group B, metformin). Endometrial biopsy was performed in all women on day 21 of the 2 simulated cycles. RESULTS: The expression of corticotropin - releasing hormone and urocortin in the endometrium was investigated. There was no significant difference between the 2 groups. A 3-day delay in the secretory maturation of the glandular epithelium relatively to the stroma was observed in 7 out of 10 women of group B (70%) as compared to only 1 out of 10 women of group A (10%, p = 0.02). CONCLUSIONS: It is shown for the first time that metformin administration to women with PCOS did not affect the expression of endometrial receptivity markers but delayed histological glandular maturation. It is suggested that metformin may have an impact on the function of the endometrium in PCOS.

Human Papilloma Virus (HPV) and Fertilization: A Mini Review.

Human papilloma virus (HPV) is one of the most prevalent viral sexually transmitted diseases. The ability of HPV to induce malignancy in the anogenital tract and stomato-pharyngeal cavity is well documented. Moreover, HPV infection may also affect reproductive health and fertility. Although, the impact of HPV on female fertility has not been thoroughly studied it has been found also to have an impact on semen parameters. Relative information can be obtained from studies investigating the relationship between HPV and pregnancy success. Furthermore, there is an ongoing debate whether HPV alters the efficacy of assisted reproductive technologies. An association between HPV and assisted reproductive technologies (ART) programs has been reported. Nevertheless, due to conflicting data and the small number of existing studies further research is required. It remains to be clarified whether HPV detection and genotyping could be included in the diagnostic procedures in couples undergoing in vitro fertilization (IVF)/intrauterine insemination (IUI) treatments. Vaccination of both genders against HPV can reduce the prevalence of HPV infection and eliminate its implications on human fertility. The aim of the present mini-review is to reiterate the association between HPV and human fertility through a systematic literature review.

Making IVF more effective through the evolution of prediction models: is prognosis the missing piece of the puzzle?

Assisted reproductive technology has evolved tremendously since the emergence of in vitro fertilization (IVF). In the course of the recent decade, there have been significant efforts in order to minimize multiple gestations, while improving percentages of singleton pregnancies and offering individualized services in IVF, in line with the trend of personalized medicine. Patients as well as clinicians and the entire IVF team benefit majorly from 'knowing what to expect' from an IVF cycle. Hereby, the question that has emerged is to what extent prognosis could facilitate toward the achievement of the above goal. In the current review, we present prediction models based on patients' characteristics and IVF data, as well as models based on embryo morphology and biomarkers during culture shaping a complication free and cost-effective personalized treatment. The starting point for the implementation of prediction models was initiated by the aspiration of moving toward optimal practice. Thus, prediction models could serve as useful tools that could safely set the expectations involved during this journey guiding and making IVF treatment more effective. The aim and scope of this review is to thoroughly present the evolution and contribution of prediction models toward an efficient IVF treatment. ABBREVIATIONS: IVF: In vitro fertilization; ART: assisted reproduction techniques; BMI: body mass index; OHSS: ovarian hyperstimulation syndrome; eSET: elective single embryo transfer; ESHRE: European Society of Human Reproduction and Embryology; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; ICSI: intracytoplasmic sperm injection; MBR: multiple birth rates; LBR: live birth rates; SART: Society for Assisted Reproductive Technology Clinic Outcome Reporting System; AFC: antral follicle count; GnRH: gonadotrophin releasing hormone; FSH: follicle stimulating hormone; LH: luteinizing hormone; AMH: anti-Müllerian hormone; DHEA: dehydroepiandrosterone; PCOS: polycystic ovarian syndrome; NPCOS: non-polycystic ovarian syndrome; CE: cost-effectiveness; CC: clomiphene citrate; ORT: ovarian reserve test; EU: embryo-uterus; DET: double embryo transfer; CES: Cumulative Embryo Score; GES: Graduated Embryo Score; CSS: Combined Scoring System; MSEQ: Mean Score of Embryo Quality; IMC: integrated morphology cleavage; EFNB2: ephrin-B2; CAMK1D: calcium/calmodulin-dependent protein kinase 1D; GSTA4: glutathione S-transferase alpha 4; GSR: glutathione reductase; PGR: progesterone receptor; AMHR2: anti-Müllerian hormone receptor 2; LIF: leukemia inhibitory factor; sHLA-G: soluble human leukocyte antigen G.

Gonadotropin Surge-Attenuating Factor: A Nonsteroidal Ovarian Hormone Controlling GnRH-Induced LH Secretion in the Normal Menstrual Cycle.

Gonadotropin surge-attenuating factor (GnSAF) is a nonsteroidal ovarian substance, which attenuates the endogenous LH surge in superovulated women. Different molecular sequences have been found, but only one of them has shown substantial homology to a known substance of the human genome. A molecular mass of 12.5kDa showing identity to the carboxyl-terminal fragment of human serum albumin and expressing GnSAF bioactivity in vitro has been identified. It has been suggested that in the normal menstrual cycle the in vivo bioactivity of GnSAF increases under the influence of the intercycle rise of FSH. GnSAF is considered the "missing link" between the ovaries and the hypothalamo-pituitary system, maintaining the pituitary in a state of low responsiveness to GnRH in the early- to midfollicular phase of the cycle. A marked decline in GnSAF bioactivity in the late follicular phase facilitates the onset and the full expression of the midcycle LH surge.

Corifollitropin alfa compared with follitropin beta in GnRH-antagonist ovarian stimulation protocols in an unselected population undergoing IVF/ICSI.

Recombinant DNA technologies have produced Corifollitropin alfa (CFa) used during IVF/ICSI in order to keep the circulating FSH levels above the threshold necessary to support multi-follicular growth for a week. In this prospective case-control study, we compared 70 participants treated with 150 µg CFa combined with 150 IU of follitropin beta (study group) with 70 subfertile participants with matching baseline characteristics, conforming with the same inclusion criteria and treated with an antagonist protocol using follitropin beta (control group). Live birth was the primary outcome, while secondary outcome measures were IVF/ICSI cycles characteristics, including adverse events and complications. Live birth was determined in reduced rates in the study compared to the control group, reaching statistical significance [6/70 versus 20/70, p = 0.002], as also in the respective number of clinical pregnancies [9/70 versus 23/70, p = 0.005], although the incidence of miscarriage was similar for both groups [6/70 versus 5/70, p > 0.99]. Most of the secondary parameters examined were similar between groups. Logistic regression revealed that protocol and AFC had a direct impact on live birth. Ovarian stimulation with CFa does not seem to constitute an equally effective method as compared with follitropin beta to be offered in a general subfertile population seeking IVF/ICSI treatments.

Submaximal doses of ghrelin do not inhibit gonadotrophin levels but stimulate prolactin secretion in postmenopausal women.

OBJECTIVE: An inhibitory effect of ghrelin on gonadotrophin secretion has been reported in normally menstruating women possibly modulated by endogenous oestrogen. The aim of this study was to examine the effect of ghrelin on gonadotrophin and prolactin (PRL) secretion in oestrogen-deprived postmenopausal women. DESIGN: Prospective intervention study. PATIENTS AND MEASUREMENTS: Ten healthy postmenopausal volunteer women were studied during two 15-days periods of oestrogen treatment (A and B) a month apart. Four experiments (Exp) were performed in total, two on day 1 (Exp 1A and Exp 1B) and two on day 15 (Exp 15A and Exp 15B) of the two periods. The women received in Exp 1A and in Exp 15A two iv injections of ghrelin (0.15 µg/kg at time 0 minute and 0.30 µg/kg at time 90 minutes) and in Exp1B and in Exp 15B normal saline (2 mL), respectively. Blood samples were taken at -15, 0, 30, 60, 90, 120, 150 and 180 minutes. RESULTS: After oestrogen treatment, late follicular phase serum oestradiol levels were attained on day 15 of periods A and B. Ghrelin administration did not affect serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), whereas it increased significantly those of growth hormone (GH) and PRL. In Exp 15A, serum PRL increment in response to ghrelin (area under the curve, net increment) was significantly greater than in Exp 1A (P<.05). CONCLUSIONS: This study demonstrates for the first time that in oestrogen-deprived postmenopausal women, ghrelin administration affects neither FSH nor LH levels but stimulates PRL secretion, that is amplified by exogenous oestrogen administration.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p < 0.05) than f-spDNA (AUC = 0.6, p > 0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

Sperm contributions to oocyte activation: more that meets the eye.

It is well known that for successful fertilization, oocyte activation is required, which involves a signal transduction cascade leading to the conversion of the oocyte to a diploid embryo. During oocyte activation, intracellular calcium levels oscillate repetitively causing exocytosis of cortical granules, the enzymes which the latter contain are released into the perivitelline space, leading to modifications of the zona pellucida (ZP), which prevent the penetration of the ZP by further spermatozoa. The necessary element that initiates oocyte activation is apparently the release of intracellular calcium (Ca(2+)) stored in the endoplasmic reticulum (ER). The exact mechanism via which Ca(2+) is released within the oocyte has not been yet clarified, and has been a matter of an ongoing debate. Today, the sperm factor hypothesis has gained general acceptance, according to which a sperm molecule, either phospholipase C (PLCζ) or a post-acrosomal sheath WW domain-binding protein (PAWP), diffuses into the ooplasm initiating a molecular cascade involving mainly the phosphoinositide pathway. Mounting evidence now indicates that these calcium oscillations are caused by a testis-specific PLC termed PLCζ, released into the oocyte following gamete fusion. Also, recently, PAWP has been proposed as an alternative sperm factor candidate. These different sperm candidates have led to a significant debate. This raises important questions as regards to the relative importance of these two proteins as diagnostic tools in reproductive medicine with therapeutic potential, indicating the need for further research. In the present mini review, the phenomenon of oocyte activation during fertilization as well as the existing controversy will be highlighted and the possible mechanisms that are involved in this process will be discussed. Finally, an explanation of the existing debate will be attempted.

Effect of Anti-Müllerian hormone (AMH) and bone morphogenetic protein 15 (BMP-15) on steroidogenesis in primary-cultured human luteinizing granulosa cells through Smad5 signalling.

PURPOSE: To determine if there is any effect of AMH and BMP-15 on estradiol and progesterone production from primary-cultured human luteinizing granulosa cells, to delineate what is the effect of FSH on their actions and which are the possible mechanisms involved. METHODS: Luteinizing granulosa cells (GCs), obtained from follicular fluid of 30 women undergoing in vitro fertilization, were cultured, after a short 24-h preincubation period, in serum-free medium for 24 or/and 48 h in the presence/absence of various concentrations of AMH, BMP-15 and FSH alone or in combinations. Estradiol and progesterone production, SMAD5 phosphorylation and StAR expression were studied in parallel. Steroids were measured in culture-supernatant using enzyme-immunoassays, while Smad5-signaling pathway activation and StAR protein expression were assessed immunocytochemically. RESULT(S): We found that the treatment of AMH in GCs for 24/48 h attenuated FSH-induced estradiol production (p < 0.001), had no effect on basal estradiol levels, decreased basal progesterone production (p < 0.001) and FSH-induced StAR expression (p < 0.001). On the other hand, BMP-15 decreased basal estradiol levels (p < 0.001) and attenuated FSH-induced estradiol production (p < 0.001). Furthermore, BMP-15 reduced progesterone basal secretion (p < 0.001), an effect that was partially reversed by FSH (p < 0.01), probably via increasing StAR expression (p < 0.001). FSH-induced StAR expression was also attenuated by BMP-15 (p < 0.001). FSH, AMH and BMP-15 activated Smad-signaling pathway, as confirmed by the increase of phospo-Smad5 protein levels (p < 0.001 compared to control). CONCLUSION(S): AMH and BMP-15 by interacting with FSH affect the production of estradiol and progesterone from cultured luteinizing-granulosa cells possibly via Smad5-protein phosphorylation.

Attenuation of the oestrogen positive feedback mechanism with the age in postmenopausal women.

OBJECTIVE: It has been reported that the positive feedback mechanism of oestrogens and progesterone is preserved, although attenuated, in late postmenopausal years. Whether this is also true for the positive feedback effect of oestrogens alone has not been investigated. DESIGN: Prospective intervention study. PATIENTS: Thirty healthy postmenopausal women. MEASUREMENTS: The women were divided into three groups according to the years since menopause (group I: 2-8 years, group II: 9-17 years, group III: 18-25 years). They were studied during a period of 41 days. Two acute experiments (EP) of exogenous oestradiol, given via skin patches, were performed from days 1 to 7 (EP1) and from days 35 to 41 (EP2) to induce an LH surge. Between the two experiments (days 7-34), oestradiol was given at the dose of 100 µg every 3 days, while oral progesterone was added from day 21 to day 34 in order to simulate a luteal phase. Blood samples were taken every 6 h during EP1 and EP2 as well as on days 8, 13, 20, 21, 27 and 34. FSH, LH, oestradiol and progesterone were measured in all blood samples. RESULTS: An LH surge occurred as a result of the oestradiol positive feedback mechanism in group I and in group II, in both EP1 and EP2. Peak LH values during the surge were significantly lower in group II than in group I in both experiments. None of the patients in group III displayed an LH surge. CONCLUSIONS: These results demonstrate for the first time a gradual attenuation of the pituitary response to oestrogenic provocation over a certain period following the menopause, with complete abolition after 20 years. It is suggested that the reserves of pituitary gonadotrophs diminish with age.

Polycystic ovaries and obesity.

Almost 50% of the women with polycystic ovary syndrome (PCOS) are obese. Obesity in PCOS affects reproduction via various mechanisms. Hyperandrogenism, increased luteinizing hormone (LH) and insulin resistance play a pivotal role. Several substances produced by the adipose tissue including leptin, adiponectin, resistin and visfatin may play a role in the pathophysiology of PCOS. Infertility in PCOS is related to anovulation. For induction of ovulation, clomiphene citrate and human gonadotrophins are first- and second-line treatments, respectively. Other treatment modalities include the use of insulin sensitizers, such as metformin as well as aromatase inhibitors and laparoscopic ovarian drilling, while in vitro fertilization is the last resort. Obesity can adversely affect infertility treatment in PCOS. Diet and lifestyle changes are recommended for the obese women before they attempt conception. The use of anti-obesity drugs and bariatric surgery in PCOS require further evaluation.