Nikos Nikolettos

Gonadotropin-releasing hormone in the ovary.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the physiology of reproduction in mammals. GnRH acts by binding to the GnRH receptor (GnRHR). In humans, only 1 conventional GnRH receptor subtype (type I GnRH receptor) has been found. In the human genome, 2 forms of GnRH have been identified, GnRH-I (mammal GnRH) and GnRH-II (chicken GnRH II). Both forms and their common receptor are expressed, apart from the hypothalamus, in various compartments of the human ovary. Gonadal steroids, gonadotropins, and GnRH itself controls the regulation of the GnRH/GnRHR system gene expression in the human ovary. The 2 types of GnRH acting paracrinally/autocrinally influence ovarian steroidogenesis, decrease the proliferation, and induce apoptosis of ovarian cells. In this review, the biology of GnRH/GnRHR system in humans, the potential roles of GnRH, and the direct effects of GnRH analogues in ovarian cells are discussed.

Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure.

Chromosomal abnormalities are thought to be responsible for implantation failure, and among chromosomal abnormalities in normally developing embryos, aneuploidy is the most frequent. Genetic testing of preimplantation embryos for chromosomal aneuploidy allows selection of chromosomally normal embryos, and early detection of chromosomal aberration will increase the chance of conceiving. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), performed by polar body or blastomere analysis, is used in infertile patients treated with assisted reproduction technologies, especially in those with a poor prognosis, e.g. repeated IVF failure, advanced maternal age, or recurrent spontaneous abortion. The aim of this paper is to clarify the impact of PGD-AS in repeated implantation failure. In this review, the data collected so far regarding PGD-AS in this patient group will be discussed in depth.

Angiogenic effect of intramuscular administration of basic and acidic fibroblast growth factor on skeletal muscles and influence of exercise on muscle angiogenesis

Background: Angiogenic factors which control the angiogenic process represent a promising strategy for restoration of blood flow, but require further evaluation before clinical use. Exercise has also been reported to induce neovascularisation in muscles. Objectives: To evaluate the angiogenic effects of basic fibroblast growth factor (b-FGF) and acidic fibroblast growth factor (a-FGF) on rat gastrocnemius muscle, when administered intramuscularly, and to compare them with those obtained by daily exercise. Methods: Forty nine rats were allotted to the following groups: A, controls; B, exercise by swimming; C1 and C2, intramuscular injection of b-FGF and a-FGF respectively; D1 and D2, b-FGF and a-FGF injection in combination with exercise. The antibody mouse anti-rat CD31 was used to evaluate the numbers of blood vessels present in histological preparations of gastrocnemius muscle. Results: Significant increases in the numbers of blood vessels of the right gastrocnemius muscles in groups C1 and D1 were observed compared with controls (p<0.05). There was only a slight increase in angiogenesis in the left gastrocnemius muscle of groups C1 and D1 compared with controls (p>0.05), and there was a decrease in angiogenesis in the gastrocnemius muscle of the swimming group compared with controls. Conclusion: The intramuscular administration of b-FGF, but not a-FGF, induced significant local angiogenesis in gastrocnemius muscle at the site of injection.

Comparison of cryopreservation outcome with human pronuclear stage oocytes obtained by the GnRH antagonist, Cetrorelix, and GnRH agonists

This retrospective study was performed to examine the implantation and pregnancy rates of frozen-thawed pronuclear stage oocytes obtained with the use of a GnRH antagonist, Cetrorelix (Cetrotide((R)) ASTA-Medica, Frankfurt/M, Germany) used in a multidose protocol with hMG, and to compare these results with those obtained after a conventional long GnRH analogue protocol (Decapeptyl-Depot, Ferring, Kiel, Germany). The study population consisted of 31 infertile couples with frozen-thawed pronuclear stage oocytes after ICSI treatment using the GnRH antagonist Cetrorelix (Cetrorelix((R))) and 31 infertile couples with frozen-thawed pronuclear stage oocytes after ICSI treatment using the long GnRH analogue protocol. Patients underwent ICSI after down regulation with a GnRH agonist (Decapeptyl) and stimulation with hMG, or a GnRH antagonist (Cetrorelix) and hMG. The supernumerary pronuclear stage oocytes were cryopreserved and transferred in a later mildly stimulated cycle. The implantation and pregnancy rates for frozen-thawed pronuclear stage oocytes derived from the GnRH antagonist compared with the GnRH agonist were 3.26% versus 3.73% (P=1.0000) and 8.33% versus 10.25% (P=1.0000), respectively. To our knowledge we report here the first pregnancies obtained by the transfer of cryopreserved pronuclear stage embryos generated from ICSI using a GnRH antagonist in the collecting cycle. The use of Cetrorelix in a multiple dose protocol in combination with hMG does not demonstrate a negative effect on viability, implantation potential or pregnancy outcome as compared to 2PN conceptuses obtained from a long GnRH agonist-hMG protocol.

Matrix metalloproteinase (MMP)-2 and MMP-9 in seminal plasma.

PurposeTo evaluate the latent and active forms of MMP-2 and MMP-9 in human semen samples and to investigate their association with semen parameters.MethodsBasic semen analysis was performed in 82 semen samples. Seminal plasma was analyzed with gelatin zymography.ResultsBoth latent and active forms of MMP-2 and MMP-9 were detected in human seminal plasma. The latent forms were the predominant ones. MMP-2 and MMP-9, either in latent or active forms, were not correlated with semen parameters. ProMMP-9 levels were higher in semen samples with abnormally low concentration (≤ 19 × 106/ml) compared with semen samples with concentration ≥ 50 × 106/ml.ConclusionMMP-2 and MMP-9 are both present in human semen. The latent forms of both MMPs are the predominant ones. ProMMP-9 is elevated in samples of low sperm concentration.

Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia

There is a lack of data regarding variables affecting the treatment outcome for non-obstructive azoospermia when spermatozoa from cryopreserved testicular specimens are utilized for ICSI. The objective of the present retrospective analysis was to investigate the effect of various parameters on treatment outcome in such cases. One hundred and sixty-five couples with non-obstructive azoospermic males undergoing a total of 297 cycles were included. In all cases the testicular tissue retrieved by multiple open-biopsy testicular sperm extraction was stored in liquid nitrogen and, after thawing, only mature spermatozoa were used for ICSI. When no motile spermatozoa were recovered, immotile spermatozoa were used. In 159 cycles, motile spermatozoa were utilized for ICSI, while in 138 cycles immotile spermatozoa were utilized. Higher normal fertilization rate (60.4 +/- 3.1 versus 51.3 +/- 1.6%, P < 0.05), number of embryos transferred (2.8 +/- 0.06 versus 2.6 +/- 0.04, P < 0.05), modified cumulative embryo score (31.2 +/- 1.6 versus 23.9 +/- 0.8, P < 0.001), and proportion of motile spermatozoa injected (67.8 versus 49.8%, P < 0.05) were observed in cycles that resulted in clinical pregnancies. Binary logistic regression analysis showed that sperm motility (odds ratio 2.06, 95% CI 1.1-3.9, P < 0.05), but not womans age, number of treatment cycle, type of GnRH-analogue used for pituitary suppression, number of oocytes retrieved or number of embryos transferred was a significant determinant of the likelihood of clinical pregnancy. In conclusion, sperm motility after freeze/thawing of testicular tissue is the major determinant of the success of ICSI in non-obstructive azoospermia.

Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia

The aim of the present study was to describe a simplified and inexpensive method of testicular tissue freezing, to assess the cumulative clinical pregnancy rate (CPR) by this technique, and to provide useful information for counselling couples with non-obstructive azoospermia. One hundred and sixty-five couples with non-obstructive azoospermic males pursuing assisted conception, from December 1995 to December 2002, were included. In all cases, the testicular tissue retrieved by open multiple-biopsy (both sides, by testicular sperm extraction) was frozen using a simple liquid nitrogen vapour freezing technique and was stored in liquid nitrogen thereafter. Only mature spermatozoa were used for intracytoplasmic sperm injection (ICSI) after thawing. Expected CPR were calculated using the Kaplan-Meier survival analysis. A total of 281 cycles were performed resulting in 53 clinical pregnancies. Crude and expected CPR (95% confidence intervals) after three cycles were 32.1 (25.7-40.1) and 55.7% (37.0-74.4) respectively. In conclusion, this simplified method for freezing testicular tissue resulted in a satisfactory outcome after ICSI in cases of non-obstructive azoospermia.

Recombinant LH in ovarian stimulation

The advent of recombinant gonadotrophins brought significant changes in fertility therapy. Treatment options with recombinant gonadotrophins add more to knowledge on folliculogenesis and ovarian steroidogenesis. Over a decade, recombinant LH (rLH) has been used for clinical trials, and the amount of peripheral LH that is necessary for optimal follicular growth, oocyte maturation, subsequent embryo development and assisted reproduction outcome during ovulation induction can now be better evaluated. This review evaluates the effect of rLH supplementation on ovarian stimulation and assisted reproduction outcome. The studies conducted with rLH supplementation in ovarian stimulation in different groups of patients and in cases of controlled ovarian stimulation are clearly discussed in this review.

ERYTHROPOIETIN ENHANCES ANGIOGENESIS IN AN EXPERIMENTAL CYCLOSPORINE A‐INDUCED NEPHROTOXICITY MODEL IN THE RAT

1 Erythropoietin (EPO) is a hormone regulating the proliferation and differentiation of erythroid precursor cells. The hypothesis that haematopoietic and endothelial cells share a common haemanglioblast progenitor among others is based on the finding that both cell lineages express cell surface antigens, such as CD31 and CD34. 2 In the present study, we investigated the angiogenic potential of recombinant human erythropoietin on cyclosporine A (CsA)‐induced nephrotoxicity in the rat kidney and compared it with the effect of basic fibroblast growth factor (bFGF), a well‐known angiogenic factor. 3 Rats were divided into five groups: A (control), B (EPO treated), C (CsA treated), D (CsA + EPO treated) and E (CsA + bFGF treated). Mouse anti‐human CD31 and CD34 antibodies were used to evaluate the kidney vessels present in histological preparations. 4 Glomerular and peritubular capillaries in Group B (EPO) were increased compared with the control (Group A; P < 0.05). Reduction of the same kidney vessels (glomerular and peritubular capillaries) in Group C (CsA; P < 0.05) compared with controls was observed, whereas in Groups D (CsA + EPO treated) and E (CsA + bFGF treated), capillaries were increased compared with Group C (CsA; P < 0.05). 5 Erythropoietin has a significant angiogenic effect in rat kidney with CsA‐induced nephrotoxicity, similar to the effect of the other angiogenic factor bFGF.

ICSI outcome in patients of 40 years age and over: a retrospective analysis

OBJECTIVE To report and analyse our experience with ICSI treatment in infertile women >/=40 years of age, with the intention of contributing to current debates on the effect of aging on the reproductive potential. STUDY DESIGN 107 infertile couples in which the female partner was aged >/=40 years and who received ICSI treatment between January 1996 and December 1998. RESULTS A total of 107 women underwent 171 treatment cycles during this period. Of 171 cycles initiated 33 were cancelled (cancellation rate=19.3%). In this way, 17 women did not have embryo transfer at all, while 90 patients had 138 cycles with oocyte retrieval and successful embryo transfer, with a mean number of embryos per transfer 2.36. Sixteen pregnancies occurred and eight of them ended in spontaneous abortion. The implantation rate was 4.9%, the pregnancy rate per initiated cycle was 9.35% and per transfer cycle 11.59%. The miscarriage rate was 50%. Moreover, 12 patients had supernumerary embryos, that were cryopreserved and transferred in 17 thawing cycles and resulted in two pregnancies ending in abortion. All pregnancies occurred when three embryos were available, except in two cases with two available embryos. The great majority of the total pregnancies (16 of 18) resulting in women aged between 40 and 42 years. CONCLUSION Our data show that women 40 and older with existing ovarian function may benefit from ICSI treatment, even when the indication for treatment is male factor infertility. Supernumerary embryos, that are cryopreserved and transferred in subsequent cycles can improve the overall pregnancy rates per oocyte retrieval, although these women should be aware of the very high risk of miscarriage.