Combination of ovarian tissue harvesting and immature oocyte collection for fertility preservation increases preservation yield.

The aim of this study was to evaluate the safety and efficacy of combined ovarian tissue cryopreservation and oocyte aspiration just before ovarian tissue cryobanking. A retrospective cohort study of fertility preservation patients treated in 2007-2013 in one tertiary centre was performed. A total of 255 cancer patients were admitted for fertility preservation: 142 patients underwent ovarian tissue cryopreservation only (OTC), 56 underwent OTC plus oocyte retrieval from ovarian tissue (OTIVM), nine underwent oocyte aspiration and in-vitro maturation (AIVM) and 48 underwent all three procedures. The total number of oocytes, total number of metaphase II (MII) oocytes, maturation rate, fertilization rate and total number of cryopreserved oocytes between groups were compared. The study found significantly more oocytes (P < 0.001), more MII oocytes (P < 0.001), better maturation rate (P < 0.01) and more cryopreserved oocytes (P < 0.05) with all three compared with OTIVM or OTC. No adverse outcome was observed by performing oocyte retrieval before ovarian resection for cryopreservation. In conclusion, oocyte aspiration just before ovarian tissue cryobanking is safe and gains more oocytes with a better maturation rate than ovarian tissue oocyte cryobanking alone. Better results were obtained with 3 days of stimulation before oocyte retrieval.

Recurrence of empty follicle syndrome with stimulated IVF cycles.

AIM: To determine the incidence of recurrent empty follicle syndrome (EFS) and to analyse the factors associated with this phenomenon. METHODS: Retrospective analysis comparing all EFS cycles with cycles in which oocytes were retrieved in our in vitro fertilization (IVF) unit between 1998 and 2006. RESULTS: Of 8292 IVF cycles, 163 (2.0%) resulted in empty follicles. Risk factors for EFS included advanced age (37.7 ± 6.0 years vs. 34.2 ± 6.0 years, p < 0.001), longer infertility (8.8 ± 10.6 years vs. 6.3 ± 8.4 years, p < 0.05), higher baseline follicle-stimulating hormone levels (8.7 ± 4.7 IU/L vs. 6.7 ± 2.9 IU/L, p < 0.001) and lower E2 levels before the human chorionic gonadotropin injection (499.9 ± 480.9 pg/mL vs. 1516.3 ± 887.5 pg/mL, p < 0.001) compared with cases in which ova were retrieved. Among patients with EFS, recurrent EFSs occurred in 15.8% of subsequent cycles. CONCLUSION: The EFS is a sporadic event in the majority of patients. However, in about 16% of the patients, EFS may recur. These cases may be a variant form of poor response and patients with repetitive EFS syndrome should be counseled concerning their chances to conceive.

IVF outcome with cryopreserved testicular sperm.

The introduction of intracytoplasmic sperm injection and the use of spermatozoa extracted from the testicles have changed the option for conception for azoospermic patients. The purpose of the present study was to evaluate the IVF outcome after using cryopreserved testicular sperm samples in comparison with fresh ones. A total of 667 in vitro fertilisation cycles with fresh or cryopreserved testicular sperm obtained by an open biopsy and testicular needle aspiration were evaluated. Sperm motility was present in 70.9% of the cycles in Group-I, 77.8% cycles in Group-II and in 83.3% In Group-III (NS). The fertilisation rates were similar in the three study groups (50%, 48.6% and 54.8% respectively). The pregnancy rates were 26.7%, 22.2% and 16.3% respectively (NS). The delivery rate, however, was significantly lower in Group-III (4.1%) than in Group-I and -II (18.4% and 15.9%, respectively, P < 0.05). The IVF results after use of cryopreserved testicular sperm are comparable with those obtained with the fresh specimens. Lack of sperm motility before cryopreservation does not exclude favourable outcome and therefore testicular sperm freezing is feasible whenever there are enough sperm cells in the extracted testicular tissue.

Does controlled ovarian stimulation prior to chemotherapy increase primordial follicle loss and diminish ovarian reserve? An animal study.

BACKGROUND: Storage of embryos for fertility preservation before chemotherapy is widely practiced. For multiple oocyte collection, the ovaries are hyperstimulated with gonadotrophins that significantly alter ovarian physiology. The effects of ovarian stimulation prior to chemotherapy on future ovarian reserve were investigated in an animal model. METHODS: Cyclophosphamide (Cy) in doses of 0, 50 or 100 mg/kg was administered to 38 adult mice (control, unstimulated). A second group of 12 mice were superovulated with equine chorionic gonadotrophin (eCG, 10 IU on Day 0) before Cy administration; hCG (10 IU) was administered (Day 2) followed by 0, 50 or 100 mg/kg Cy (Day 4). In both groups ovaries were removed, serially sectioned (7-day post-Cy), primordial follicles were counted and differences between groups evaluated. RESULTS: Follicle number dropped from 469 +/- 24 (mean +/- SE) to 307 +/- 27 and 234 +/- 19 with 50 or 100 mg/kg Cy, respectively (P < 0.0001). In the eCG pretreated group, follicle count dropped from 480 +/- 31 to 345 +/- 16 and 211 +/- 26 when 50 or 100 mg/kg Cy were administered (P < 0.0001). There were no significant differences in follicle count between the pretreated eCG group and controls for each chemotherapy dose. CONCLUSIONS: This animal study indicates that ovarian stimulation before administration of Cy does not adversely affect ovarian reserve post-treatment. These results provide support for the safety of fertility preservation using ovarian stimulation and IVF-embryo cryopreservation procedures prior to chemotherapy.

A study of the knowledge, attitudes and practices of Tibetan yak herders with respect to brucellosis.

Background: Little information is available regarding the knowledge, attitudes and practices (KAP) of the owners of yaks in Tibet with respect to brucellosis. Methods: This manuscript reports on the results of a cross-sectional survey using a structured questionnaire investigating the KAP of 317 yak herders from 34 villages in the three main yak-rearing counties of Maizhokunggar, Damxung and Yadong from April to August 2015. Results: Although 60.6% of the respondents had heard of brucellosis, there was an overall low level of knowledge about the disease. Herders did, however, adopt management/husbandry practices which would reduce transmission of the disease to humans and other animals. Multivariable logistic modelling showed that a better knowledge was predicted by age, production system adopted, education level and number of people in a household. Differences in attitudes and practices were predicted by education level, production system and household size. Conclusions: In order to increase the awareness of herders on brucellosis and to reduce the risk of infection in both humans and other animals, public health education campaigns should be developed and offered to livestock owners in Tibet.

A cytosolic protein tyrosine kinase activity is induced by follicle stimulating hormone and insulin like growth factor-I in human granulosa cells.

Follicle stimulating hormone (FSH) induces estradiol (E2) production in rat, porcine and human granulosa cells with a concomitant increase in cAMP. In human granulosa cells insulin like growth factor-I (IGF-I) induces E2 production without cAMP accumulation. In the current study we report that IGF-I and FSH effects on aromatase activity both involve activation of a cytosolic soluble protein tyrosine kinase (CytPTK). This FSH and IGF-I stimulated CytPTK activity was blocked by AG-82 (a tyrosine kinase inhibitor) and by staurosporine (STS) (a non specific protein kinase inhibitor) at concentrations which inhibited E2 production. These new findings strengthen the concept of fail-safe mechanism in E2 production in human granulosa cells by an involvement of tyrosine kinase(s) activity downstream of cAMP formation and protein kinase A (PKA) activation.

Steroid secretion by granulosa cells isolated from a woman with 17 alpha-hydroxylase deficiency.

Steroid synthesis in luteal/granulosa cells harvested after follicular aspiration in a patient with 17 alpha-hydroxylase deficiency was investigated. Follicular stimulation with purified FSH after the suppression of ACTH and gonadotropin secretion by corticoid analogs and superactive GnRH agonist permitted ovum pick-up and the study of steroid synthesis in the obtained granulosa cells. Progesterone synthesis was elevated while 17 alpha-hydroxy progesterone, testosterone, androstenedione, and estrogen production was minimal. Aromatase activity was retained in these cells, as demonstrated by the 100-fold increase in estrogen levels after the addition of androgens. Progesterone levels in follicular fluids were in the normal range, while estrogen, 17 alpha-hydroxy progesterone, and androgen levels were minimal. These results provide in vitro evidence of 17 alpha-hydroxylase deficiency in the granulosa cells of this patient.

Insulin-like growth factor-I and follicle-stimulating hormone suppress insulin-like growth factor binding protein-1 secretion by human granulosa-luteal cells.

Local regulation of insulin-like growth factor binding protein-1 (IGFBP-1) production in the human ovarian follicle was investigated using cultured human granulosa-luteal cells. Both insulin-like growth factor-I (IGF-I) and follicle-stimulating hormone (FSH) exerted a dual effect on granulosa cells: while estradiol (E2) production was increased by both stimulants, the addition of either of the two hormones led to a reduction in IGFBP-1 secretion by more than 50%. Inhibition of IGFBP-1 production in response to IGF-I was dose-dependent,with the highest effect observed at 5 nM IGF-I. A significant correlation was found between the increase in E2 and inhibition of IGFBP-1 secretion in response to IGF-I. These observations may suggest a novel mechanism, at the follicular level, by which FSH and IGF-I amplify the IGF-I effect in the ovarian follicular cells.

Insulin-like growth factor-I (IGF-I) may not be essential for ovarian follicular development: evidence from IGF-I deficiency.

Insulin-like growth factor-I (IGF-I) stimulates growth and differentiation in follicular granulosa cells (GC). To examine whether this effect is prerequisite to human folliculogenesis, a patient with Laron-type dwarfism (IGF-I deficiency secondary to GH receptor abnormality) was examined while undergoing in vitro fertilization treatment. Despite low levels of IGF-I in serum and follicular fluid (less than 3 and less than 2 nmol/L) and very high levels of IGF-I-binding protein, the patient developed normal ovarian follicles. After the administration of GnRH analog (GnRHa) and human menopausal gonadotropin in a dose similar to that used in normovulatory women, estradiol (E2) levels reached above 5000 pmol/L on the day of hCG administration, and mature fertilizable oocytes were retrieved during ovum pickup. The patient's GC E2 production, tested in a primary culture, did not respond to IGF-I after 4 days of incubation, while control cultures showed a significant increase. Only after a priming period of 7 days did IGF-I have a significant effect on E2 production, as observed in the patient's GC culture. This delayed response suggests that the patient's GC were not exposed in vivo to IGF-I. Our data support the view that IGF-I is not required for normal follicular development, but is, rather, a nonessential modulator of FSH action.

Studies on sperm chromosomes in patients with severe male factor infertility undergoing assisted reproductive technology treatment.

The aim of the study was to determine the rate of chromosome abnormalities in testicular sperm after intracytoplasmic sperm injection due to severe male factor infertility. The study groups included patient with non-obstructive azoospermia (n=9), obstructive azoospermia (n=10), Klinefelter's syndrome (n=5) and normal controls (n=6, groups I-VI, respectively). The mean serum levels of FSH 17.5+/-8.2 (P<0.05), 3.5+/-2.6, 29.8+/-13.0 (P<0.05) and 3.1+/-0.4 mIU/ml, respectively. The rates of chromosome abnormalities were 19.6% (P<0.001), 8.2% (P<0.001), 6.3 and 1.6%, respectively. Chromosomes X and Y were significantly more involved in the aneuploidy than chromosome 18 in groups I and II. The present findings demonstrate a linkage between gonadal failure (high serum FSH levels) and sperm chromosome abnormalities. Our findings may explain the increased incidence of perinatal sex chromosome abnormalities found in severe male factor patients. Patients with non-mosaic Klinefelter's syndrome have comparable risk for sex chromosomes aneuploidy as the rest of the patients with azoospermia. Therefore, genetic screening during pregnancy or before embryo replacement should be carefully considered in severe male factor patient following in vitro fertilization (IVF).

Protocols for induction of ovulation. The concept of programmed cycles.

Programmed oocyte retrieval which includes suppression of the hypothalamic-pituitary-ovarian axis by oral contraceptives or GnRH analogue and predetermined ("fixed") day for ovum pick-up has been demonstrated to yield a pregnancy rate that is comparable to that achieved by the conventional individualized approach to follicular maturation. We have prospectively compared two regimens of suppression (the pill and GnRH analogue) followed by two ovarian stimulation protocols (clomiphene citrate + human menopausal gonadotropin [CC + hMG] and pure follicle-stimulating hormone + human menopausal gonadotropin [pure FSH + hMG]). Sixteen patients were studied in each group. It was found that the latent phase, which represents a period of ovarian insensitivity, was prolonged and directly correlated to the duration of suppression, and that suppression with the GnRH analogue was associated with a shorter latent phase than that with the pill. Suppression with the pill for 30 days compared with 15 days resulted in a greater cancellation of laparoscopic oocyte retrieval, a lower fertilization rate, and a lower pregnancy rate. The numbers of oocytes recovered, fertilized, and cleaved were similar in both stimulation protocols. The use of the GnRH analogue for 30 days compared with 15 days was associated with a lower pregnancy rate, even though the number of oocytes that were recovered and fertilized were similar. Ovarian stimulation with pure FSH + hMG resulted in a shorter latent phase than did stimulation with CC + hMG, but the results of treatment with both protocols were similar. It is concluded that both the pill and GnRH analogue are acceptable means of manipulating the cycle and the day of oocyte retrieval. However, they should be used for the shortest periods possible because prolonged use is associated with some unwarranted effects. For each suppression-stimulation protocol there seems to be a different fixed day for retrieval that should be established prospectively.

Conception rate after gonadotropin therapy in hyperprolactinemia and normoprolactinemia.

The cumulative pregnancy rate after gonadotropin treatment was evaluated in 63 hyperprolactinemic and 242 normoprolactinemic women. All pregnancies in the hyperprolactinemic patients were achieved within four treatment cycles; the cumulative pregnancy was 62% as compared with 29% in normoprolactinemics. The same results were obtained when patients were divided according to endogenous estrogenic activity. These results imply that in bromocriptine failures there is no need to lower prolactin levels to achieve pregnancy with gonadotropins.

Triplet pregnancy in uterus didelphys with delivery interval of 72 days.

A unique case of triplet pregnancy in a woman with uterus didelphys is described, with fetuses A and B in the right uterine horn and fetus C in the left horn. Missed abortion of fetus A was diagnosed in the 22nd week of gestation by real-time ultrasonography, but only in the 27th week of gestation did the right uterine horn contract, resulting in expulsion of fetus A. Fetus B was delivered by cesarean section 2 hours later. Fetus C remained in the left uterine horn until the 37th week of gestation, when a viable 2490-g female infant was delivered by cesarean section. Triplet pregnancy occurs with sufficient frequency to be unremarkable; however, triplet pregnancy in a patient with uterus didelphys and with 72 days' lapse between the delivery of the first 2 fetuses and delivery of the third is a rare occurrence.

An evaluation of etiologic factors and therapy in 665 infertile couples.

Six hundred and sixty-five unselected infertile couples were investigated and treated in a private clinic during a 15-year span. Male infertility factors were involved in 28%, ovulatory disturbances in 31.5%, tubal factors in 16.3%, and undiscovered factors in 17.6% (117 couples). In 50 couples (7.5%) fertility-reducing factors were found in both partners, with a 14% pregnancy rate, compared with a 54.3% pregnancy rate when only a female factor was involved. The chances of successful treatment of females decline steadily with age, correlating with the fact that the longer the exposure time before the start of treatment, the less the chance of pregnancy. Investigation following 1 year of exposure should be advised. Pregnancy rates were 50% in ovulatory disturbances and 30% with tubal factors. Predictably, a progressive increase in the pregnancy rate was noted during the span of this study.

Antisperm antibodies: their effect on the process of fertilization studied in vitro.

The influence of antisperm antibodies on the fertilizing capacity of human sperm was investigated in a zone-free hamster egg system using heterologous and homologous antisera. The ability of these antisera to interfere with the fertilizing capacity of human spermatozoa correlated well with the antibody titers obtained using the Franklin-Dukes tube slide test. However, there was little correlation between the antifertility effects of these antisera and their activities in the gelatin and tray agglutination tests. The addition of complement to the incubation medium amplified the inhibitory activity exhibited by these antisera, although the extent of this inhibitory influence bore no relationship to the antibody titers obtained using the complement-dependent sperm-immobilization technique. The study demonstrates the importance of the hamster egg penetration test in evaluating the antifertility effect of antisperm antibodies.

Ischemic stroke due to induction of ovulation with clomiphene citrate and menotropins without evidence of ovarian hyperstimulation syndrome.

Cerebral infarction due to menotropin therapy without evidence of ovarian hyperstimulation is presented. The patient was successfully treated with aspirin and anticonvulsive agents and was discharged without residual neurologic symptoms.

Treatment-independent pregnancy after in vitro fertilization and embryo transfer trial.

This study indicates that SP occur in a higher percentage than theoretically expected after IVE-ET treatment, but it is not conclusive as to whether the SP is independent or a result of the treatment or operative laparoscopy. Our results are in agreement with those of previous studies that have shown that SP occur with almost all kinds of infertility. We therefore reject the previous suggestion of Steptoe, who argued that to prevent ectopic pregnancy from complicating embryo replacement, preparatory electrocauterization of diseased tube should be performed. We also suggest that as long as the results of treatment are below the normal monthly spontaneous conception rate, there is justification for a prospective random study that includes all diagnoses other than BTB. Such a study might help make any future selection of patients for treatment more appropriate.

Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated failure of implantation.

OBJECTIVE: To evaluate the efficacy of zygote intrafallopian transfer (ZIFT) on implantation rates and pregnancy rates (PRs) in patients with repeated failure of implantation in IVF-ET cycles. DESIGN: A case-control study. PATIENT(S): Criteria for patient selection included male factor or unexplained infertility, normal uterine cavity, and at least three failures of implantation in IVF-ET cycles in which at least three embryos were placed per transfer. Data on 70 patients who underwent 92 ZIFT cycles are presented. A control group consisted of patients with the same selection criteria who underwent an additional standard IVF-ET cycle during the same time period. INTERVENTION(S): Ovulation induction consisted of down-regulation with GnRH analogue followed by ovarian stimulation with FSH and hMG. Intracytoplasmic sperm injection was performed on the oocytes of all patients with male factor infertility. Zygotes were transferred by laparoscopy into the fallopian tube 24-26 hours after oocyte retrieval. MAIN OUTCOME MEASURE(S): Implantation rates and PRs in the ZIFT and control groups were compared. RESULT(S): The PRs and implantation rates were significantly higher in the ZIFT group than in the control group: 34.2% (24/70) and 8.7% (29/333) versus 17.1% (12/70) and 4.4% (13/289), respectively (P = 0.002 and P = 0.04). The cumulative conception rate for two ZIFT cycles was 59.3%. CONCLUSION(S): Zygote intrafallopian transfer should be considered a beneficial mode of treatment for patients with repeated failure of implantation in IVF and transcervical ET. More prospective randomized studies are needed to support this observation.

Chromosome analysis of multipronuclear human oocytes fertilized in vitro.

In an in vitro fertilization and embryo transfer program composed mainly of patients with tubal infertility, the incidence of fertilized oocytes with multiple pronuclei was 4.3%. An attempt was made to fix such oocytes with supernumerary pronuclei in order to assess the chromosome constitution of the gametes. Nine multipronuclear oocytes containing a total of 29 pronuclei were successfully fixed before the first cleavage division, and another oocyte containing 3 pronuclei was fixed before the second cleavage division. On analysis, chromosome counts could be obtained for 28 of the 29 total pronuclei, and 24 gave informative results. Nineteen pronuclei had a normal haploid chromosome count, 2 pronuclei contained one extra chromosome, and 3 pronuclei had one chromosome missing. The presence of a Y chromosome in six pronuclei identified their paternal origin; two of the six sperm pronuclei had a 22,Y,-E chromosome constitution. Only three pronuclei could be conclusively ascertained to be maternal in origin, yet two of these were aneuploid; one pronucleus had a 24,X,+D karyotype and the second had only 22 chromosomes. Multipronuclear oocytes present ideal material for analyzing the chromosome constitution of those human gametes which can undergo fertilization in vitro.

Complete failure of fertilization in couples with unexplained infertility: implications for subsequent in vitro fertilization cycles.

OBJECTIVE: To determine whether complete failure of fertilization of oocytes in couples with unexplained infertility persists during subsequent in vitro fertilization (IVF) cycles. DESIGN: A retrospective study of 120 cycles of IVF in 44 couples with unexplained infertility and complete failure of fertilization during their first IVF trial. SETTING: In vitro fertilization unit of the Sheba Medical Center. PATIENTS: Forty-four couples undergoing IVF for unexplained infertility. MAIN OUTCOME MEASURE(S): Fertilization rate of retrieved oocytes and pregnancy rate. RESULTS: Of the initial 44 couples, 37 underwent additional IVF cycles and 30 (81.1%) achieved fertilization. Seven patients conceived during the study period as a result of IVF and two conceived spontaneously. CONCLUSIONS: Complete failure of fertilization does not necessarily persist during subsequent IVF cycles. Therefore, at least two more IVF cycles should be attempted before reverting to other therapeutic options. However, although fertilization can be achieved in most couples, the mean oocyte fertilization rate during subsequent cycles in this group is low. This suggests an underlying undiagnosed pathology of oocyte/sperm interaction in some of these patients.