Use of granulocyte colony-stimulating factor for the treatment of unexplained recurrent miscarriage: a randomised controlled trial.

BACKGROUND: Recurrent miscarriage (RM) is defined as the occurrence of three or more clinically detectable pregnancy losses in the first trimester. In most cases of RM, its aetiology remains unexplained. Granulocyte colony-stimulating factor (G-CSF), a cytokine, and its receptor are expressed in placental tissue. To investigate the effectiveness of G-CSF in preventing embryo demise, we administered G-CSF to women with RM. METHODS: A randomised controlled trial in women with RM treated with G-CSF or placebo was conducted in one private reproductive medicine clinic. Sixty-eight women with unexplained primary RM, all with at least four consecutive miscarriages and negative for all clinical investigations, were selected. Patients were randomized for s.c. treatment with G-CSF (n = 35) (1 microg/kg/day) starting on the sixth day after ovulation, or with placebo (n = 33). Patients were randomized using a computer-generated randomization number sequence. Pregnancy outcome (delivery of a healthy baby without major or minor malformations) was the primary outcome measure. RESULTS In the group treated with G-CSF, 29 out of 35 (82.8%) women delivered a healthy baby, whereas in the placebo group, this figure was only 16 out of 33 (48.5%) (P = 0.0061, odds ratio = 5.1; 95% confidence interval 1.5-18.4). Significantly higher beta-hCG levels were found in gestation weeks 5-9 in women treated with G-CSF versus placebo (P < 0.001). CONCLUSIONS: Our data show that G-CSF may be effective in the treatment of unexplained RM. However, further studies are needed to confirm the effectiveness of this treatment in women with unexplained RM, refractory to conventional treatment. The study was registered with a ICMJE recognized registry, the Clinical Trial.gov Protocol Registry System, with the number NCT00772122.

Short gonadotropin-releasing hormone agonist versus flexible antagonist versus clomiphene citrate regimens in poor responders undergoing in vitro fertilization: a randomized controlled trial.

OBJECTIVE: Poor responders represent a frustrating condition for couples undergoing IVF and clinicians, and their treatment remains disputed. To assess the efficacy and the most suitable protocol, we conducted a randomized controlled trial comparing three different protocols of ovarian stimulation in poor responder women: clomiphene citrate (CC) plus a high dose of gonadotropins and GnRH antagonist, flexible GnRH antagonist protocol and a short GnRH agonist protocol. PATIENTS AND METHODS: Between July 2014 and December 2015 we enrolled 250 poor responders in a previous IVF cycle at least 3 months before. We divided into three groups: group A, 68 women treated with clomiphene citrate and FSH plus antagonist; Group B, 71 patients treated with FSH plus antagonist; Group C, 75 patients treated with FSH plus GnRH agonist. RESULTS: The GnRH agonist protocol showed a significantly higher pregnancy rate (29.3% vs. 5.9% vs. 14.1% respectively) than the clomiphene and the GnRH antagonist protocol, number of mature oocytes collected, estradiol levels and endometrial thickness. The cost of medications for each baby born was lower for the GnRH agonist protocol than for the others; the implantation rate was significantly lower in the clomiphene group (4.8%) than in the GnRH antagonist group (9.3%) and the GnRH agonist groups (19.2%). No significant differences emerged for total FSH administered, days of stimulation, numbers of oocytes retrieved and embryos transferred. CONCLUSIONS: This study demonstrates that short GnRH agonist protocol should be the first choice in poor responders; instead, clomiphene citrate should be avoided due to its very low success rate and high costs.

Sperm function and choice of preparation media: comparison of Percoll and Accudenz discontinuous density gradients.

We compared the sperm populations prepared by Accudenz (35-65%) and Percoll (40-80%) density gradients in 21 normospermic specimens (concentration, 53.6 +/- 3.8 x 10(6) sperm/ml; motility, 44.5 +/- 3.5%). Accudenz facilitated a higher recovery of sperm and motile sperm (68.4 +/- 6.6% vs. 49.3 +/- 4.9%, P < 0.001, and 87.8 +/- 4.1% vs. 77.8 +/- 3.7%, P < 0.01, respectively). Sperm motility was lower in the Accudenz compared to the Percoll pellets; thus the values of total motile sperm recovered were not different (17.1 +/- 2.4 vs. 15 + /- 1 2.2 x 10(6) sperm/ml). The long term retention of sperm motility was substantially improved in Accudenz (at 24 hours, 34.9 +/- 2.8% vs. 26.3 +/- 1.5%; 60% vs. 40% of the initial motility, P < 0.001), and the Accudenz vs. Percoll samples also exhibited a higher retention of total motile sperm (at 24 hours, 9.8 +/-.2 vs. 6.1 = 0.5 x 10(6) motile sperm/ml, P < 0.05). The sperm motility index, a multiple of velocity and motility in the sample that reflects the efficiency of the sperm population in sperm-oocyte interaction, was 75% higher in the Accudenz samples at 24 hours (3.6 +/- 0.4 vs. 2.1 +/- 0.2, mu m/second, P < 0.01). Sperm cellular maturity by the creatine phosphokinase (CK) activity and CK-M to CK-B isoform ratio parameters (in the original samples 0.14 +/- 0.02 lU CK/100 x 10(6) sperm and 57.9 +/- 3.7%, respectively) were improved in both the Accudenz and Percoll pellets (P < 0.001), with no difference between the two sperm fractions. Sperm activation status monitored by chlortetracycline fluorescence indicated that after 4 hours of incubation the incidence of fully acrosome-reacted spermatozoa in the Accudenz versus Percoll pellets was 6.2 +/- 0.3% versus 13.1 +/- 1.0% (P < 0.001), a 100% increase in Percoll. We can conclude that Accudenz yields a higher concentration of motile spermatozoa, with improved retention of motility, velocity, and acrosomal integrity and without an increase of sperm with diminished cellular maturity. Thus, in sperm preparation for intrauterine insemination, in which the timing of ovulation and insemination frequently do not correspond, Accudenz-prepared sperm, with a better retention of motility/velocity and acrosomal integrity and with a consequential higher resistance to activation by the female reproductive tract, are expected to be more effective.

Serum CA 125 and first trimester abortion.

OBJECTIVE: The study was carried out to assess the clinical value of serum CA 125 in association with serum beta-human chorionic gonadotropin (hCG) for predicting pregnancy outcome. METHODS: One hundred spontaneous pregnancies, 52 non-threatened pregnancies and 48 with threatened abortions, observed in the Department of Obstetrics and Gynecology at the University 'La Sapienza', Rome, Italy, were evaluated during the first trimester using ultrasound examination, CA 125 and beta-hCG titrations. RESULTS: Threatened pregnancies had statistically significantly higher CA 125 serum values than non-threatened pregnancies, especially those with a negative outcome (P < 0.01). The CA 125 levels in the threatened pregnancies were positively correlated with the tropho-decidual hematoma volume (r=0.839, P < 0.0001). The CA 125-beta-hCG association showed a higher prognostic value (sensitivity 78.9%, specificity 96.5%) in assessing pregnancy outcome than CA 125 or 0-hCG alone (sensitivity 78.9% and 57.9%, respectively; specificity 75.8% and 86.2%, respectively). CONCLUSIONS: Our findings are in accordance with the hypothesis of a tropho-decidual origin of this marker, suggesting its possible usefulness in the prognostic evaluation of first trimester threatened abortion.

Serum luteinizing hormone, follicle-stimulating hormone and oestradiol pattern in women undergoing pituitary suppression with different gonadotrophin-releasing hormone analogue protocols for assisted reproduction.

Gonadotrophin-releasing hormone analogues (GnRH-a) are used widely in controlled ovarian stimulation (COS) cycles for assisted reproduction. At present, there is great debate about the influence of exogenous hormone activity on the hypothalamus-pituitary axis following pituitary desensitization. The objective of this comparative study was to investigate the pattern of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol in women undergoing ovarian stimulation with different GnRH-a preparations. We retrospectively analysed 201 women, aged between 27 and 43 years, who were referred consecutively to our infertility clinic between January 2002 and January 2003. All women had no endocrinopathies or occult ovarian failure as assessed by day-3 hormone profile. Women were enrolled in one of the following COS protocols: depot triptorelin long protocol (n = 38), buserelin long protocol (n = 101) or buserelin short protocol (n = 62). Recombinant FSH was used to induce ovulation. Treatment was monitored by transvaginal ultrasound scan and serum measurement of FSH, LH and oestradiol. Among the women initially included, 30 had cancelled cycles due to poor ovarian response. Serum LH levels were significantly higher in the short-protocol group compared with the long-protocol groups (p < 0.001). The number of follicles, oocyte yield, number of grade-I embryos and fertilization rate were significantly lower in the short-protocol group than in the long-protocol groups. These findings showed that LH concentrations are significantly higher in women undergoing reversible medical hypophysectomy with a GnRH-a short protocol than in women treated with a long protocol. The hypothesis of an LH ceiling is confirmed.

HLA antigen sharing in Italian couples in which women were affected by gestational trophoblastic tumors.

PROBLEM: The development of gestational trophoblastic tumors (GTT), in which genetic factors are strongly involved, is a rare event. To test the possibility that gene(s) linked to the Major histocompatibility Complex (MHC) may have a role in both embryo growth and tumor development, the HLA typing was performed on patients affected by GTT and on their partners. METHOD: The study group of sixteen couples, in which the women were affected by an invasive mole or choriocarcinoma, and the control group of thirty normal fertile couples without history of spontaneous abortion or GTT were typed for class I and class II HLA antigen. RESULTS: The results showed no differences in single HLA-A and B antigen frequency between GTT couples and controls. In HLA-DR, locus an increased frequency of DR-6 antigen was observed (p < 0.05). No differences were observed in the frequency of number of antigens shared. When considering the single locus no differences were found in the sharing of the antigens of the A and B locus, while the frequency of antigenic sharing for DR locus was significantly higher in GTT couples with respect to controls (p < 0.025). Furthermore a higher frequency of Bw35-DR5 antigenic combination was found in GTT partners than in controls (P < 0.02). CONCLUSIONS: These data represent a confirmation of the existence of a MHC linked gene(s) influencing the GTT development.

Serum lipoperoxide level variations in normal and luteal phase defect cycles.

A luteal phase defect (LPD) is a clinical problem leading to infertility and is characterized by reduced progesterone levels during the luteal phase. Serum lipoperoxidation is an expression of cellular hypoxic damage occurring under all stress-oxidative conditions. Forty-eight consecutive infertility patients, according to the infertility class II of the WHO classification, underwent clinical observation. Twenty-five patients ovulated constantly with a normal luteal phase, while the other 23 had a LPD, hormonally and histologically assessed. The luteal phase was monitored biochemically by titration of progesterone, oestradiol, and serum lipid peroxides during the ovulatory period and 6-12 days after ovulation. The mean plasmatic concentration of oestradiol was not statistically different between the LPD and the normal-cycle groups, while a statistically significant difference was found for progesterone plasmatic concentrations between the LPD and the normal-cycle groups (p < 0.05); a greater statistical difference was also found in the mean serum lipoperoxide titration (p < 0.001). These findings suggest that serum lipoperoxidation titration may be a useful tool in the study of the interactions between hormonal milieu and sympathetic activity.

Influence of histocompatibility antigens in recurrent spontaneous abortion couples and on their reproductive performances.

PROBLEM: To determine if human leukocyte antigens (HLA) play any role in the aetiology of recurrent spontaneous abortion (RSA), a substantial group of RSA couples were studied, and their reproductive performances in a 3-year follow-up recorded. METHODS: HLA typing was performed for HLA-A, -B, and DR antigens in both partners of 75 couples with unexplained RSA, and compared with a control group of 30 fertile couples that never experienced abortion. A further 57 couples of this group were studied for their reproductive performance in a 3-year follow-up, and subdivided into three subgroups: 1) couples that achieved successful pregnancy during the follow-up, and subdivided into three subgroups: 1) couples that achieved successful pregnancy during the follow-up; 2) couples that experienced abortion and no livebirth during the follow-up; and 3) couples that experienced infertility during the follow-up. RESULTS: There were no significant differences for antigen frequency in all the different HLA loci, and HLA antigen sharing between all the RSA couples and controls. Significant increase of sharing for HLA-DR locus was observed in the couples that aborted during the follow-up with respect to the couples that achieved livebirth and controls (P < 0.03 and P < 0.02 respectively), and significantly increased frequency of B44, DR5 antigen combination in the same comparison (P < 0.03). No significant differences were observed in terms of the interval between conceptions in couples without antigen sharing with respect to couples with 1, 2 or more antigens shared, and antigen sharing in Locus A, B or DR. CONCLUSIONS: The results suggest that gene(s) disadvantageous for reproduction may exist between the HLA-B and -DR chromosomal region which influences the pregnancy outcome in RSA couples, and that HLA-antigen sharing itself does not influence the outcome.

Is local anesthesia an affordable alternative to general anesthesia for minilaparoscopy?

STUDY OBJECTIVE: To determine if minilaparoscopy under local anesthesia is at least as reliable and affordable as that performed under general anesthesia. DESIGN: Prospective, randomized study (Canadian Task Force classification I). SETTING: University-affiliated hospital. PATIENTS: One hundred sixty-four consecutive women evaluated for infertility. INTERVENTION: Diagnostic minilaparoscopy performed after women were randomized to receive general or local anesthesia with conscious sedation. MEASUREMENTS AND MAIN RESULTS: Levels of postoperative pain measured by visual analog scale; volume of CO(2) used; length of procedure, complete pelvic evaluation, and hospitalization; complications; and pathologic diagnosis were evaluated. The groups were comparable in age, years of infertility, and symptoms. For women receiving local anesthesia, 5.5% required general anesthesia to complete the procedure. Women in both groups required postoperative analgesics. The groups had no statistically significant differences in pain level 1 hour after the procedure, number of complications, and pelvic pathology. Patients who had local anesthesia required a smaller volume of CO(2) (p <0.01) and their hospitalization was significantly shorter (p <0.01). However, in 15% of these women pelvic visualization was incomplete, compared with 7.2% in the general anesthesia group. CONCLUSION: Minilaparoscopy performed under local anesthesia was as reliable and affordable as when performed under general anesthesia.

Efficacy of biochemical vs biological monitoring during the "timing" of the chorionic gonadotropin administration in FSH and HMG stimulated cycles.

The problem of an exact timing of the HCG administration in stimulated cycles (for infertility problems) is of primary importance for optimizing the results. In this study the authors show the results obtained in two groups of patients to whom HCG was administered according to two different methods of monitoring: the first biochemical, the second biological. The examination of the results has proven that the insertion of the biological method (cervical mucus) among those used in the monitoring has allowed a significant increase of the biological response to treatment.

Hyaluronic acid substantially increases the retention of motility in cryopreserved/thawed human spermatozoa.

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.

Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios.

Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

Preferential location of sex chromosomes, their aneuploidy in human sperm, and their role in determining sex chromosome aneuploidy in embryos after ICSI.

BACKGROUND: In babies born after ICSI procedures, an increase of de-novo sex chromosome abnormalities has been observed. Several hypotheses have been proposed to explain these findings: an increased rate of sex chromosome aneuploidy in sperm of oligozoospermic men, or a preferential location of the sex chromosomes in the sub-acrosomal region of the sperm nucleus which leads to a reduced DNA decondensation of this region. In order to investigate which theory may be more reliable, we studied the localization of sex chromosomes and their aneuploidy rate in sperm in men undergoing ICSI. METHODS: Using fluorescent in-situ hybridization we studied sex chromosome localization and the aneuploidy rate for sex chromosomes and chromosome 18 in 20 oligospermic men undergoing ICSI and in 10 controls. RESULTS: In 40.94 and 52.92% of cases, the X and Y chromosomes respectively were localized in the sub-acrosomal region of the sperm nucleus compared with only 14.29% of cases of chromosome 18 (P < 0.001). An increase of sex chromosome aneuploidy in sperm of oligospermic men was observed; 2.91 versus 0.69% of controls (P < 0.001). CONCLUSIONS: Sex chromosomes are localized preferentially in the sub-acrosomal region of sperm and sex chromosome aneuploidy rate in the sperm of oligozoospermic men is higher than in controls.

Blood levels of lipids, lipoperoxides, vitamin E and glutathione peroxidase in women with habitual abortion.

Plasma levels of triglycerides, cholesterol, cholesterol esters, phospholipids, lipoperoxides, vitamin E and erythrocyte glutathione peroxidase activity showed no significant differences between 40 women with habitual abortion and controls. However, the levels of free fatty acids (FFA), which are extremely cytotoxic compounds, were significantly higher in women with habitual abortion (HA) than in controls (16.8 +/- 6.7 vs. 8.6 +/- 3.7 mg/100 ml, p < 0.01, respectively). The high amounts of FFA in HA women during pregnancy were probably due to a continuous and/or excessive stress-dependent discharge into the blood of catecholamines from autonomic nerve endings. These catecholamines can induce a strong uterine vasoconstriction and placental ischemia-hypoxia which, in association with additional insults caused by reoxygenation, might lead to eventual miscarriage.

Arterial and venous Doppler velocimetry in the severely growth-restricted fetus and associations with adverse perinatal outcome.

OBJECTIVE: To evaluate the association between arterial and venous Doppler waveforms and adverse perinatal outcome in severe intrauterine growth restriction. DESIGN: Fetuses between 26 and 32 weeks' gestation with ultrasonographically estimated fetal weight below the 5th centile were included in this prospective study. The last Doppler measurements of the umbilical artery, ductus venosus, umbilical vein and middle cerebral artery before delivery and adverse outcome parameters without gestational age independence were used for statistical analysis. Adverse outcome parameters were defined as the following: fetal demise or neonatal death in the first 30 days of life; 5-min Apgar score of < 7; intubation at birth; retinopathy of prematurity; respiratory distress syndrome; necrotizing enterocolitis; stay in neonatal intensive care unit of more than 60 days; intraventricular hemorrhage; periventricular leukomalacia. RESULTS: Nineteen fetuses fulfilled the inclusion criteria. Perinatal death, low 5-min Apgar values and necrotizing enterocolitis were gestational age-independent adverse outcome parameters. Abnormal Doppler velocimetry of the ductus venosus was the only significant parameter associated with perinatal death and low 5-min Apgar scores. None of the Doppler parameters predicted necrotizing enterocolitis. CONCLUSION: Our data suggest that management of severely growth-restricted fetuses may be aided by the study of ductus venosus Doppler velocimetry.

Successful pregnancies following an egg donation program in women with previously treated Hodgkin's disease.

BACKGROUND AND OBJECTIVES: In order to draw attention not only to patients affected by a neoplasia, but also to those who may have problems of sterility, we describe six women affected by Hodgkin's disease who had precocious menopause due to chemotherapy and/or radiotherapy but who were safely delivered of children. These pregnancies were achieved through oocyte donation, in vitro fertilization and intrauterine embryo transfer or oocyte intracytoplasmic insemination. DESIGN AND METHODS: During natural or iatrogenic menopause, the uterus preserves its capacity to respond to steroidal hormones and to permit implantation and development of an embryo. Our study concerns six young females with iatrogenic menopause caused by treatment of Hodgkin's disease who carried a pregnancy to term. The pregnancies were achieved by oocyte donation, in vitro fertilization and intrauterine embryo transfer or oocyte intracytoplasmic insemination. Endometrial maturation was obtained by administration of estradiol and progesterone. Steroidal therapy was administered until the 13th-14th week in relation to placental function. RESULTS: Five of the 6 females underwent Caesarean section because of a twin birth or complications during the third trimester of pregnancy (gestosis). All the delivered children are, to date, well; their median age is 4 years. INTERPRETATION AND CONCLUSIONS: This study confirms the possibility of women treated for Hodgkin's disease being able to carry a pregnancy safely to term with the help of steroidal therapy. Careful clinical and obstetric surveillance is important. Focusing attention on long-term survivors of Hodgkin's disease, we set the goal of improving the quality of life of these patients, considering their psychophysical well-being as a whole. Greater attention to the problems of safeguarding fertility in these patients would be advisable, also in the light of legislative regulation of medical care techniques in various countries.

Pain mapping during minilaparoscopy in infertile patients without pathology.

STUDY OBJECTIVE: To draw a map of pelvic pain and quantify the level of provoked pain during minilaparoscopy under local anesthesia and conscious sedation. DESIGN: Observational study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: Twenty infertile women. INTERVENTIONS: Minilaparoscopy was performed under local anesthesia and conscious sedation, and cognitive performance was evaluated with the Rey auditory verbal learning task. MEASUREMENTS AND MAIN RESULTS: The diagnostic procedure was performed with one 2-mm micrograsper and one 2-mm microprobe to evaluate the pelvis. In particular we grasped utero-ovarian ligaments; we touched, grasped, and distended fallopian tubes with blue dye; we moved the uterus with a manipulator inserted at the cervix; and we touched and grasped bowel and omentum. Level of pain was recorded on a visual analog scale. Patients had no pathologic findings, including minimal endometriosis and pelvic adhesions. The highest level of pain was recorded when we distended the tubes. No pain was elicited when we touched and grasped ovary, omentum, and bowel. In 10% of women when we stretched the tubo-ovarian ligament we provoked a minimal vagal reaction. CONCLUSION: Minilaparoscopy under conscious sedation for pelvic pain mapping in women without pain or pathology revealed consistently negative findings, validating the value of this measurement. (J Am Assoc Gynecol Laparosc 6(1):51-54, 1999)