Modification of peripheric Treg and CD56brightNK levels in RIF women after egg donation, treated with GM-CSF or placebo.

Recurrent implantation failure (RIF) is defined as when implantation repeatedly failed to reach a stage recognizable by pelvic ultrasound in IVF cycle and it may be due to several causes. The GM-CSF is a cytokine promoting leukocyte growth and trophoblast development: we tested it to treat these patients in a pilot-controlled trial evaluating the modification of peripheric Treg and CD56brightNK levels after the treatment with this cytokine and in control patients affected by RIF after egg donation cycles. This study was performed on 24 RIF women after egg donation cycles. Single good quality blastocyst transfer was performed in the cycle object of this study. Patients were randomly assigned to two groups: 12 women treated with subcutaneous GM-CSF 0.3 mg/kg/daily from the day before embryo transfer to the ß-hCG day, and 12 women treated with subcutaneous saline solution infusion as control. All patients were tested for Treg and CD56brightNK cell levels in blood circulation before and after treatment using flow-cytometry with specific antibodies. The two groups of patients were similar for epidemiologic characteristics, the ongoing pregnancy rate in the GM-CSF group was 83.3% whereas in the control group was 25.0% (P = 0.0123). In the study group there was a significative increase of Treg cells (P < 0.001) with respect to the levels before treatment and to control group. Instead, the levels of CD56brightNK did not show any significative variation. Our study showed that the treatment with GM-CSF increases the Treg cells in the peripheric blood.

Immunohistochemical Study on the Expression of G-CSF, G-CSFR, VEGF, VEGFR-1, Foxp3 in First Trimester Trophoblast of Recurrent Pregnancy Loss in Pregnancies Treated with G-CSF and Controls.

BACKGROUND: Recurrent Pregnancy Loss (RPL) is a syndrome recognizing several causes, and in some cases the treatment with Granulocyte Colony Stimulating Factor (G-CSF) may be successful, especially when karyotype of the previous miscarriage showed no embryo chromosomal abnormalities. In order to evaluate the effects of G-CSF treatment on the decidual and trophoblast expression of G-CSF and its receptor, VEGF and its receptor and Foxp3, specific marker of putative Tregs we conducted an immunohistochemical study. METHODS: This study was conducted on three groups of patients for a total of 38 women: in 8 cases decidual and trophoblast tissue were obtained from 8 women with unexplained RPL treated with G-CSF that miscarried despite treatment; in 15 cases the tissue were obtained from 15 women with unexplained RPL no treated; 15 cases of women who underwent voluntary pregnancy termination were used as controls. Tissue collected from these patients were used for immunohistochemistry studies testing the expression of G-CSF, G-CSFR, VEGF, VEGFR-1 and Foxp3. RESULTS: G-CSF treatment increased the concentration of cells expressing Foxp3, specific marker for Tregs, in the decidua, whereas in no treated RPL a reduction of these cells was found when compared to controls. Furthermore, G-CSF treatment increased the expression of G-CSF and VEGF in the trophoblast. CONCLUSIONS: Our study showed that G-CSF treatment increased the number of decidual Treg cells in RPL patients as well as the expression of G-CSF and VEGF in villus trophoblast. These finding may explain the effectiveness of this treatment in RPL, probably regulating the maternal immune response through Tregs recruitment in the decidua, as well as stimulating trophoblast growth.

PreImplantation Factor in endometriosis: A potential role in inducing immune privilege for ectopic endometrium.

Endometriosis is a chronic inflammatory condition characterised by the growth of endometrial epithelial and stromal cells outside the uterine cavity. In addition to Sampson's theory of retrograde menstruation, endometriosis pathogenesis is facilitated by a privileged inflammatory microenvironment, with T regulatory FoxP3+ expressing T cells (Tregs) being a significant factor. PreImplantation Factor (PIF) is a peptide essential for pregnancy recognition and development. An immune modulatory function of the synthetic PIF analog (sPIF) has been successfully confirmed in multiple animal models. We report that PIF is expressed in the epithelial ectopic cells in close proximity to FoxP3+ stromal cells. We provide evidence that PIF interacts with FoxP3+ cells and modulates cell viability, dependent on cell source and presence of inflammatory mediators. Our finding represent a novel PIF-based mechanism in endometriosis that has potential for novel therapeutics.

Ovarian Stimulation Protocol in IVF: An Up-to-Date Review of the Literature.

The Assisted Reproductive Technology (ART) was born in order to help couples with infertility issues in having a baby. The first treatments of IVF used the spontaneous cycle of the women, with the retrieval of only one oocyte. Further studies have shown that it is possible to induce ovulation by administrating gonadotropins during the menstrual cycle, in order to obtain a higher number of oocytes. Many stimulation protocols have been introduced for controlled ovarian hyperstimulation of patients undergoing in vitro fertilization treatment. This review describe the different stimulation protocols using follicle-stimulating hormone (FSH) in combination with Gonadotropin releasing hormone (GnRH) either agonist or antagonist, oral supplementations and ovarian triggering. Using GnRH antagonist protocols have been demonstrated to improve significantly the clinical pregnancy rates for expected poor and high-responders, and in those women at high risk of developing ovarian hyperstimulation syndrome (OHSS). Two meta-analyses showed a better outcome in terms of the live birth rate when highly purified human menopausal gonadotropin (HMG) was used for ovarian stimulation compared with recombinant follicle stimulating hormone (rFSH) in the GnRH agonist long protocol. One of the most efficient stimulation protocol is the use of a combined protocol of human derived urinary FSH (uFSH) and rFSH. Combined protocol has resulted in a significant increase in the proportion of mature metaphase II oocytes and grade 1 embryos when compared to either rFSH or uFSH alone. A significantly higher delivery rate was achieved in rFSH+uFSH compared to the other protocols in poor and normal responders. Studying the combination of melatonin with myo-inositol and folic acid has also showed a higher percentage of mature oocytes in the melatonin group and a higher percentage of G1 embryos as well. However, It remains a crucial step to confirm the efficacy of such protocols for clinical application and it is still needs to comparison studies on larger scale with more focused on the differences in patients' response criteria and additional confounding variables, in order to draw more defined conclusions.

Fas and Fas-Ligand in Eutopic and Ectopic Endometrium of Women With Endometriosis: The Possible Immune Privilege of Ectopic Endometrium.

The Fas/Fas-Ligand system is an important mediator of apoptosis. We analyzed their expression in tissue specimens obtained from 33 women with severe endometriosis and 18 women without endometriosis. Immunostaining for Fas-Ligand in the eutopic endometrium was stronger in the epithelial cells of secretory phase, while the epithelial cells of endometriotic lesions showed a significantly stronger staining for Fas-Ligand independently from the menstrual phase (P < 0.01). Immunostaining for Fas in the eutopic endometrium showed a reduced staining during the proliferative phase, whereas it was strong in the secretory phase. The epithelial cells of the ectopic endometrium showed a reduced staining for Fas independently from the menstrual phase with respect to the eutopic tissue (P < 0.01). The reduced expression of Fas in the ectopic endometrium with the contemporary higher expression of Fas-Ligand in the corresponding cells suggests a possible immune privilege of this tissue.

Expression of oct-4 and c-kit antigens in endometriosis.

The objective of this study was to test the expression of the oct-4 and c-kit, both markers of stem cells, in the ectopic endometrial tissue of endometriotic lesions of women with severe endometriosis. Our findings show that ectopic epithelial cells express oct-4 and c-kit and this suggests that the ectopic endometrium in endometriosis has a stem cell origin and could explain the possible progression to ovarian cancer.

Clinical complications after transvaginal oocyte retrieval in 7,098 IVF cycles.

We report the complications observed after transvaginal oocyte retrieval guided by ultrasound in 7,098 IVF cycles. The frequency of severe complications in our patients was 0.08%, of which four cases were intraperitoneal bleeding (0.06%) and two were cases of ovarian abscess (0.003%).

Urinary hMG (Meropur) versus recombinant FSH plus recombinant LH (Pergoveris) in IVF: a multicenter, prospective, randomized controlled trial.

To compare IVF outcome in ovarian stimulation protocols with recombinant FSH plus recombinant LH versus hMG, 122 patients were randomized into two study groups: group A, patients treated with urinary hMG, and group B, patients treated with rFSH plus rLH. The two groups proved to be comparable to the main IVF outcome (pregnancy rate, implantation rate, oocytes, and embryos quality), with an increasing risk of ovarian hyperstimulation in the Pergoveris group.

Cetrorelix protocol versus gonadotropin-releasing hormone analog suppression long protocol for superovulation in intracytoplasmic sperm injection patients older than 40.

OBJECTIVE: To determine which protocols work better between cetrorelix and long protocols in older patients in a randomized controlled study. DESIGN: A controlled randomized study in a single private IVF center. SETTING: Infertile women referred to a private IVF center. PATIENT(S): Five hundred sixty-four women 40 years or older undergoing IVF. INTERVENTION(S): At their first IVF cycle, the women were randomized into two study groups using a computer-generated number sequence: 281 cases were treated with the cetrorelix protocol, and 283 patients were treated with a long protocol for controlled ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Days of stimulation, E(2) on the day of hCG administration, amount of FSH administered, number of oocytes yielded, number of embryos obtained, pregnancy rate, and implantation rate. RESULT(S): Patients treated with the long protocol showed a significantly higher number of oocytes retrieved and a higher pregnancy rate for both the cycle and transfer with respect to the cetrorelix protocol patients. The other parameter evaluated did not show any statistically significant differences. CONCLUSION(S): Our study showed that the long protocol performed better in older women than the cetrorelix protocol and that the GnRH antagonist may be detrimental in older women.

Autoimmune response to Chlamydia trachomatis infection and in vitro fertilization outcome.

This observational study was conducted in 235 patients undergoing IVF who had a cervical swab positive for Chlamydia trachomatis and who underwent antibiotic treatment until a negative cervical swab before IVF attempt. After oocyte retrieval, follicular fluids of 109 patients out of 228 still showed the presence of IgA antichlamydia antibodies and a significantly lower pregnancy and implantation rate; therefore we conclude that patients should undergo IVF procedure after serum antichlamydia IgA tests negative.

Natural-cycle in vitro fertilization in poor responder patients: a survey of 500 consecutive cycles.

OBJECTIVE: To determine the role of the natural cycle for in vitro fertilization (IVF) in poor responder patients. DESIGN: Retrospective survey. SETTING: Private center for assisted reproduction. PATIENT(S): 294 women who were poor responders in a previous IVF cycle. INTERVENTION(S): Analysis of 500 consecutive natural cycles IVF. MAIN OUTCOME MEASURE(S): Number of cycles with oocytes, pregnancy rate per cycle, per transfer, and implantation rate. RESULT(S): Oocytes were found in 391 cases (78.1%), and cleaving embryos suitable for transfer were obtained in 285 cycles (57.0%). Pregnancy was observed in 49 cases, with a pregnancy rate of 9.8% per cycle, 17.1% per transfer, and 16.7% per patient. The patients were subdivided arbitrarily by the women's age into three groups. Patients 35 years old or younger showed a pregnancy rate of 18.1% per cycle, 29.2% per transfer, and 31.7% per patient. Women aged between 36 and 39 years showed a pregnancy rate of 11.7% per cycle, 20.6% per transfer, and 20.3% per patient. Women 40 years old or older showed a pregnancy rate of 5.8% per cycle, 10.5% per transfer, and 9.7% per patient. No differences were found for any of the evaluated parameters, independent of which cycle was the first, the second, third, fourth, or fifth, or further consecutive cycle. CONCLUSION(S): In poor responder patients, natural-cycle IVF is an effective treatment, especially in younger women.

Myolysis of uterine fibroids: is there a role?

Myolysis is among the new procedures under development for the treatment of symptoms related to uterine leiomyoma. The procedure targets the destruction of fibroids using one of a number of focused energy delivery systems including those based upon radiofrequency electricity, supercooled cryoprobes, and, most recently, focused ultrasound monitored by real time magnetic resonance imaging. For thermomyolysis and cryomyolysis, delivery of the energy requires access to the tissue by laparoscopy, and, in some instances, hysteroscopy. For focused ultrasound, the patient is detached from the energy source, which is delivered by an array of external beams. Clinical evaluation has been confined to case series, but it is evident that the approach results in a variable degree of reduction of the total uterine mass, and, usually, a reduction in uterine bleeding. Clearly, longer term appropriately designed comparative trials are required that evaluate and compare myolysis with myomectomy, uterine artery embolization, and hysterectomy, to name a few.

Urinary follicle-stimulating hormone (FSH) is more effective than recombinant FSH in older women in a controlled randomized study.

OBJECTIVE: The following study was conducted to determine which FSH, recombinant or urinary, works better in older women. DESIGN: We conducted a controlled randomized study in a single university IVF center. SETTING: University IVF center. PATIENT(S): Women (N = 257) over 39 years old undergoing IVF. INTERVENTION(S): The patients were randomized into two study groups at their first IVF cycle: 121 patients were treated with recombinant FSH, and 120 patients were treated with urinary FSH. Both groups were suppressed with a long GnRH analog protocol. MAIN OUTCOME MEASURE(S): Days of stimulation, E2 at the day of hCG, total amount of FSH administered, number of oocytes collected, amount of FSH per oocyte, and number of embryos obtained. RESULT(S): Patients treated with urinary FSH required a significantly lower total amount of FSH, and a lower amount of FSH per oocyte than women treated with recombinant FSH. The other measures evaluated did not show any statistically significant differences. CONCLUSION(S): Our study showed that urinary FSH performed better in older women than recombinant FSH when associated with the long protocol.

Use of a medium buffered with N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) in intracytoplasmic sperm injection procedures is detrimental to the outcome of in vitro fertilization.

OBJECTIVE: This study was conducted to determine whether N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES)-buffered medium used for the microinjection of sperm into oocytes may be detrimental for the embryo. DESIGN: Controlled randomized study. SETTING: Private IVF center. PATIENT(S): Women (n = 708) undergoing ICSI. INTERVENTION(S): The women were randomized into two study groups: 2,204 oocytes from 357 women were treated using a medium buffered with bicarbonate without HEPES during the ICSI procedure, and 2,168 oocytes from 351 women were treated using a medium buffered with HEPES during the ICSI procedure. MAIN OUTCOME MEASURE(S): Fertilization rate, degeneration rate, triploid rate, cleavage rate, embryo quality, pregnancy rate, implantation rate, and abortion rate. RESULT(S): Oocytes treated with a HEPES-buffered medium showed a statistically significant higher rate of triploid and degenerated oocytes after fertilization with ICSI compared with oocytes treated with a medium without HEPES. The embryos obtained from oocytes microinjected with a HEPES-buffered medium showed a statistically significant higher rate of highly fragmented embryos compared with the controls. Pregnancy rate and implantation rate were statistically significantly lower in the patient group with oocytes treated with the HEPES-buffered medium. The other parameters evaluated did not show any statistically significant differences. CONCLUSION(S): Our study showed that the use of media buffered with HEPES, during the microinjection of sperm into the oocytes, is detrimental for IVF outcome and should be avoided.

Short versus long gonadotropin-releasing hormone analogue suppression protocols for superovulation in patients > or = 40 years old undergoing intracytoplasmic sperm injection.

OBJECTIVE: To determine whether the short or long protocol for controlled ovarian hyperstimulation works better in older patients undergoing IVF. DESIGN: Controlled, randomized study. SETTING: A single private IVF center. PATIENT(S): Two hundred twenty infertile women aged > or = 40 years undergoing IVF. INTERVENTION(S): At their first IVF cycle, the women were randomized into two study groups according to a computer-generated number sequence: 110 patients were treated with a long protocol, and the other 110 were treated with a short protocol for controlled ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Days of stimulation, E2 level at the day of hCG administration, amount of FSH administered, number of oocytes collected, number of embryos obtained, pregnancy rate, implantation rate. RESULT(S): Patients treated with a long protocol showed a significantly higher number of oocytes retrieved, a higher number of embryos obtained, and a higher pregnancy rate, both for cycle and transfer, compared with the short-protocol patients. The other parameters evaluated did not show any statistically significant differences. CONCLUSION(S): Our study showed that the long protocol performed better than the short protocol in older women. Our findings demonstrated that flare-up in older women might be detrimental.

Directed laparoscopic cryomyolysis for symptomatic leiomyomata: one-year follow up.

STUDY OBJECTIVE: To evaluate the long-term effectiveness of laparoscopic cryomyolysis as a minimally invasive technique for the treatment of symptomatic uterine myomas in menstruating women. DESIGN: Open, one-arm pilot study (Canadian Task Force classification II). SETTING: University-affiliated public hospital. PATIENTS: Twenty patients with symptomatic uterine myomas were treated with directed cryomyolysis. All had reported abnormal bleeding and/or pelvic pain/pressure and/or urinary frequency. Myoma diameters varied from 4 to 10 cm. INTERVENTION: One-year follow-up after laparoscopic-directed cryomyolysis. MEASUREMENTS AND MAIN RESULTS: Laparoscopic cryomyolysis was performed using the Her Option Cryoablation Unit (American Medical Systems, Minneapolis, MN). Patients were evaluated 1, 3, 6, 9, and 12 months after surgery. Power color Doppler ultrasound was performed preoperatively and postoperatively to demonstrate the effectiveness of the technique in reducing or eliminating the primary blood supply to the myomas, as well documenting regression of the myomas. All patients reported a high rate of satisfaction with the treatment including absence of symptoms 12 months after surgery, with no bleeding and no myoma-related symptoms, comparable with patients who underwent hysterectomy. Mean shrinkage of myoma volume increased until 9 months after surgery (59.5% +/- 13.2%), reaching a steady mean-volume reduction of approximately 60% (61.9% +/- 11.9%) 12 months after surgery. CONCLUSIONS: Directed laparoscopic cryomyolysis appears to be an effective and safe technique for providing rapid symptom relief and at least 12 months' effectiveness in the treatment of symptomatic uterine leiomyomas.

Add-back therapy in the treatment of endometriosis-associated pain.

OBJECTIVE: To determine the efficacy of GnRH analogue plus add-back therapy compared with GnRH analogue alone and estroprogestin in patients with relapse of endometriosis-associated pain. DESIGN: Randomized, controlled study. SETTING: University hospital. PATIENT(S): One hundred thirty-three women with relapse of endometriosis-related pain after previous endometriosis surgery. INTERVENTION(S): Forty-six women were treated with GnRH analogue plus add-back therapy, 44 women were given GnRH analogue alone, and 43 women received estroprogestin, for 12 months. MAIN OUTCOME MEASURE(S): Pain evaluation by a visual analogue scale, quality of life in treated patients according to the SF-36 questionnaire, and occurrence of adverse effects, including bone mass density loss, at pretreatment, after 6 months of treatment, at the end of treatment (12 months), and 6 months after discontinuation of treatment. RESULT(S): Patients treated either with GnRH analogue alone or GnRH analogue plus add-back therapy showed a higher reduction of pelvic pain, dysmenorrhea, and dyspareunia than patients treated with oral contraceptive, whereas patients treated with add-back therapy showed a better quality of life, as assessed with the SF-36 questionnaire, and adverse effects rate than the other two groups. CONCLUSION(S): Add-back therapy allows the treatment of women with relapse of endometriosis-associated pain for a longer period, with reduced bone mineral density loss, good control of pain symptoms, and better patient quality of life compared with GnRH analogue alone or oral contraceptive.

A controlled trial of natural cycle versus microdose gonadotropin-releasing hormone analog flare cycles in poor responders undergoing in vitro fertilization.

OBJECTIVE: To determine the efficacy of natural-cycle IVF compared with controlled ovarian hyperstimulation in poor responders. DESIGN: Randomized, controlled study. SETTING: Private center for assisted reproduction. PATIENT(S): One hundred twenty-nine women who were poor responders in a previous IVF cycle. INTERVENTION(S): Fifty-nine women underwent 114 attempts of natural-cycle IVF, and 70 women underwent 101 attempts of IVF with controlled ovarian hyperstimulation with microdose GnRH analog flare. MAIN OUTCOME MEASURE(S): Number of oocytes retrieved, pregnancy rate (PR) per cycle, PR per transfer, and implantation rate. RESULT(S): The poor responders treated with natural-cycle IVF and those treated with micro-GnRH analog flare showed similar PRs per cycle and per transfer. The women treated with natural-cycle IVF showed a statistically significant higher implantation rate (14.9%) compared with controls (5.5%). When subdivided into three groups according to age (or=36-39 years, >or=40 years), younger patients had a better PR than the other two groups. CONCLUSION(S): In poor responders, natural-cycle IVF is at least as effective as controlled ovarian hyperstimulation, especially in younger patients, with a better implantation rate.