Walter Ciampaglia

Physiologic ICSI: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality.

OBJECTIVE To evaluate the role of hyaluronic acid (HA) for sperm selection before intracytoplasmic sperm injection (ICSI). DESIGN Three prospective studies. SETTING Private assisted reproduction center in Italy. PATIENT(S) Study 1: 20 men. Study 2: 15 men. Study 3: 206 couples treated with ICSI on a limited number of oocytes per patient (1-3) in accordance with Italian IVF law. INTERVENTION(S) Study 1: determination of sperm DNA fragmentation of HA-bound spermatozoa versus spermatozoa in polyvinylpyrrolidone (PVP). Study 2: assessment of nuclear morphology of HA-bound spermatozoa versus spermatozoa in PVP. Study 3: randomized study comparing conventional PVP-ICSI to ICSI in which the spermatozoa are selected for their capacity to bind to HA (HA-ICSI). MAIN OUTCOME MEASURE(S) Study 1: sperm DNA fragmentation rate. Study 2: sperm nucleus normalcy rate according to motile sperm organellar morphology examination criteria. Study 3: fertilization, embryo quality and development, and implantation and pregnancy. RESULT(S) Spematozoa bound to HA show a significant reduction in DNA fragmentation (study 1) and a significant improvement in nucleus normalcy (study 2) compared with spermatozoa immersed in PVP. Furthermore, injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and development (study 3). CONCLUSION(S) Hyaluronic acid may optimize ICSI outcome by favoring selection of spermatozoa without DNA fragmentation and with normal nucleus. Furthermore, HA may also be used to speed up the selection of spermatozoa with normal nucleus during intracytoplasmic morphologically selected sperm injection (IMSI).

Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes.

The present study reports, as far as is known for the first time, the safety of UV sterilization of liquid nitrogen and hermetical cryostorage of human oocytes by comparing the efficiency of fresh and vitrified sibling oocytes of infertile patients. A prospective randomized study on sibling oocytes of 31 patients was carried out. Metaphase-II oocytes were randomized for intracytoplasmic sperm injection and the supernumerary sibling oocytes were vitrified using a novel Cryotop aseptic procedure (UV liquid nitrogen sterilization and hermetical cryostorage). After unsuccessful attempts with fresh oocytes, vitrified sibling oocytes were injected. Mean outcome measures observed were fertilization, cleavage and top-quality embryo rates. No significant differences were observed between the fresh and vitrified-warmed sibling oocytes: oocyte fertilization was 88.3% versus 84.9%; cleavage 72.6% versus 71.0%; top-quality embryos 33.8% versus 26.3% and mean number of transferred embryos 2.6 ± 0.1 versus 2.5 ± 0.1, respectively. Clinical pregnancy rate per cycle with vitrified-warmed oocytes was 35.5% (implantation rate 17.1%) and seven healthy babies were born. This study demonstrated that UV liquid nitrogen sterilization and hermetical cryostorage does not adversely affect the developmental competence of vitrified oocytes, allowing safe aseptic open vitrification applicable under strict directives on tissue manipulation.

Efficiency of hyaluronic acid (HA) sperm selection

PurposeHyaluronic Acid (HA) has a role as “physiologic selector” for spermatozoa prior to intracytoplasmic sperm injection (ICSI). The objective of this study is to analyze the results achievable by the introduction of a routine HA-ICSI programme.MethodsWe retrospectively observed 293 couples treated with HA-ICSI versus 86 couples treated with conventional PVP-ICSI (historical control group). ICSI was performed on a limited number of oocytes per patient (1–3) according to Italian IVF law at the time of the study. Main outcome measures observed were: fertilization, embryo quality, implantation and pregnancy.ResultsThis study showed that Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation.ConclusionsIf wider multi-center randomized studies will confirm these beneficial effects on ICSI outcome, HA could be considered as a routine choice for “physiologic” sperm selection prior to ICSI.

Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration

BACKGROUND A number of factors influence the success of oocyte cryopreservation and subsequent ICSI. The aim of the present study is to establish the ideal time, after oocyte retrieval, for human oocyte cryopreservation via slow freezing/rapid thawing protocol with 0.3 M sucrose concentration in cryoprotectant solution (SF/RT 0.3 M). METHODS Retrospective study with 75 patients on the clinical outcome of 93 oocyte thawing cycles divided into three groups. Group A: freezing within 2 h from oocyte retrieval. Group B: freezing between 2 and 3 h from retrieval. Group C: freezing after 3 h. RESULTS The rate of best quality embryos was significantly higher (35.2%; P = 0.050) in Group A than in Group C (14.1%). Pregnancy and implantation rates were 39.1% (9/23) and 18.5% (10/54) in Group A. Nine clinical pregnancies per 124 thawed (7.3%) and 73 injected (12.3%) oocytes were observed in Group A versus 3 pregnancies per 174 thawed, 103 injected (1.7%, 2.9%, P = 0.046 and 0.0049) in Group B and 4 per 139 thawed, 88 injected (2.9%, 4.5%, NS) in Group C. The overall yield from oocytes cryopreserved within 2 h of retrieval was 8.1 implantations per 100 oocytes thawed. CONCLUSIONS Embryo quality and clinical outcome of thawing cycles were significantly improved when oocyte cryopreservation by SF/RT 0.3 M was carried out within 2 h from oocyte retrieval.

Heterogeneity in the responsiveness to long-term lifestyle intervention and predictability in obese women with polycystic ovary syndrome

BACKGROUND Treatment of obesity improves all features of the polycystic ovary syndrome (PCOS). There is, however, a heterogeneous response to weight loss, and predictive factors are unknown. Objective This follow-up study aimed to investigate obese women with PCOS treated with a long-term lifestyle program to evaluate responsiveness and predictability. METHODS One hundred PCOS women meeting the criteria for selection were invited to participate and 65 of them agreed. Lifestyle intervention had consisted of a 1200-1400 kcal/day diet for 6 months, followed by mild calorie restriction and physical activity. The protocol, which was similar at baseline and follow-up, included anthropometry, clinical evaluation, pelvic ultrasound, and laboratory investigations. The mean follow-up period was 20.4±12.5 months. RESULTS After the follow-up period, women were reclassified into three groups according to the persistence (group 1, 15.4%), partial (group 2, 47.7%), or complete (group 3, 36.9%) disappearance of the categorical features of PCOS (hyperandrogenism, menses, and ovulatory dysfunctions). Duration of the follow-up and extent of weight loss were similar among the three groups, as were fasting and glucose-stimulated insulin and indices of insulin resistance. Baseline waist circumference, waist to hip ratio (WHR), and androstenedione blood levels were negatively correlated with a better outcome in the univariate analysis. However, only basal androstenedione values persisted to a highly significant extent (P<0.001) in the multivariate analysis. CONCLUSIONS Responsiveness to weight loss in overweight/obese PCOS women varies considerably and more than one third of women may achieve full recovery. These findings add new perspectives to the impact of obesity on the pathophysiology of PCOS.

Low-dose human chorionic gonadotropin therapy can improve sensitivity to exogenous follicle-stimulating hormone in patients with secondary amenorrhea

OBJECTIVE To assess the effect of supplementing an ovulation induction regimen of highly purified FSH with LH activity in the form of low-dose hCG therapy. DESIGN Case report. SETTING The Reproductive Endocrinology Center at the University of Bologna, Bologna, Italy. PATIENT(S) A woman with weight-related secondary hypogonadotropic amenorrhea. INTERVENTION(S) The patient was treated first with highly purified FSH alone and then received highly purified FSH in combination with low-dose hCG therapy (50 IU/d). MAIN OUTCOME MEASURE(S) Pelvic ultrasound examinations, serum E2 levels, duration of treatment, total dose of highly purified FSH, and outcome of treatment. RESULT(S) The concomitant administration of low-dose hCG and highly purified FSH markedly reduced the duration of treatment and the dose of highly purified FSH, and resulted in a quadruplet pregnancy in a patient in whom several previous ovulation induction procedures had been unsuccessful. CONCLUSION(S) Supplementation of an ovulation induction regimen with an agent that has LH activity can enhance FSH-induced folliculogenesis and markedly reduce costs in women with hypogonadotropic hypogonadism. However, this increased response can be associated with complications such as multiple gestation.

Current concepts and novel applications of LH activity in ovarian stimulation

Luteinizing hormone (LH) is a crucial physiological regulator of the human menstrual cycle. LH activity is also contained in many medications used to treat anovulation and to stimulate multiple folliculogenesis for assisted reproduction techniques. However, LH activity had previously been regarded as just a contaminant of follicle-stimulating hormone (FSH)-containing products and deemed potentially detrimental for reproductive function. Novel experimental and clinical evidence now suggests that the administration of pharmacological amounts of LH activity, instead of being harmful, is therapeutically advantageous, particularly in the support and modulation of ovarian folliculogenesis. The aim of this article is to provide an overview of the effects of LH activity administration in ovarian stimulation and to outline novel unconventional gonadotropin regimens that might improve the efficacy, safety and convenience of ovulation induction.

Intracytoplasmic sperm injection pregnancy after low-dose human chorionic gonadotropin alone to support ovarian folliculogenesis

OBJECTIVE To prove that several days of low-dose hCG alone can be used to stimulate folliculogenesis, complete FSH-initiated follicle/oocyte maturation, and achieve pregnancy in assisted reproduction technology. DESIGN Case report. SETTING Reproductive endocrinology center at an academic institution. PATIENT(S) A 35-year-old female patient and her partner with male-related infertility. INTERVENTION(S) After an 8-day priming with hMG (225 IU/d), we administered low-dose hCG (200 IU/d) alone for 5 days in one GnRH-agonist suppressed patient until proper follicle development was obtained and intracytoplasmic sperm injection was performed. MAIN OUTCOME MEASURE(S) Daily serum levels of LH, FSH, hCG, E(2), P, and T; measurements of follicle number and size; oocytes retrieved and fertilized; pregnancy. RESULT(S) Although FSH levels rapidly declined after hMG discontinuation, E(2) and large follicles increased during hCG-only administration. Several good quality oocytes were retrieved and fertilized by intracytoplasmic sperm injection; three embryos were transferred and a twin pregnancy ensued. CONCLUSION(S) Replacement of FSH with low-dose hCG for several days in the late ovulation induction stages of assisted reproduction technology resulted in: [1] continued growth of large ovarian follicles and E(2); [2] an optimal preovulatory follicle pattern consisting of many large and few medium and small follicles; and [3] reproductively competent oocytes and pregnancy.

Increased clearance of cortisol by 5β-reductase in a subgroup of women with adrenal hyperandrogenism in polycystic ovary syndrome

Objective: Increased peripheral metabolism of cortisol may explain compensatory ACTH-dependent adrenal steroidogenesis and hence hyperandrogenism in polycystic ovary syndrome (PCOS). Previous studies have described an increased 5α-reduction of cortisol or impaired regeneration of cortisol by 11β-HSD1 in PCOS. However, these observations may be confounded by obesity. Moreover, the relationship between alterations in cortisol metabolism and the extent of adrenal androgen hyper-secretion in response to ACTH has not been established. This study aimed to examine the association between cortisol metabolism and ACTH-dependent adrenal hyperandrogenism in PCOS, independently of obesity.Design: We compared 90 PCOS women (age 18–45 yr) stratified by adrenal androgen responses to ACTH1–24 and 45 controls matched for age and body weight. Methods: PCOS women were stratified as normal responders (NR), intermediate responders (IR), and high responders (HR) to 250 ug ACTH1–24: NR (no.=27) had androstenedione and DHEA responses within 2 SD of the mean in controls; IR (no.=43) had DHEA responses >2 SD above controls; HR (no.=20) had both androstenedione and DHEA responses >2 SD above controls. Results: All groups were similar for age, body weight, and body fat distribution. Basal testosterone, androstenedione, and 5α-dihydrotestosterone plasma levels were similarly elevated among the 3 groups of PCOS compared with controls, whereas basal DHEA-S was higher in HR (2.8±1.2 μg/ml) and IR (2.4±1.1 μg/ml) than in NR (1.8±0.8 μg/ml) and controls (1.7±0.6 μg/ml). The HR group had the lowest basal plasma cortisol levels (101±36 ng/ml vs IR 135±42 ng/ml, NR 144±48 ng/ml, and controls 165±48 ng/ml; all p<0.01), but the greatest cortisol response to ACTH1–24 (Δ(60–0)cortisol 173±60 ng/ml vs IR 136±51 ng/ml, NR 114±50 ng/ml, and controls 127±50 ng/ml; all p<0.01), and the highest urinary excretion of total and 5β-reduced cortisol metabolites (eg 5β-tetrahydrocortisol/cortisol ratio 25.2±15.3 vs IR 18.8±10.7, NR 19.7±11.4, and controls 17.2±13.7; all p<0.05). There were no differences in urinary excretion of 5α-reduced cortisol metabolites or in 5α-dihydrotestosterone/testosterone ratio between groups. Conclusions: Adrenal androgen excess in PCOS is associated with increased inactivation of cortisol by 5β-reductase that may lower cortisol blood levels and stimulate ACTH-dependent steroidogenesis.

Basal insulin-like factor 3 levels predict functional ovarian hyperandrogenism in the polycystic ovary syndrome

Aim: The aims of the study were to understand the association between insulin-like factor 3 (INSL3) and functional ovarian hyperandrogenism (FOH) in PCOS and the regulatory role played by LH. Subjects and methods: Fifteen PCOS women were classified as FOH (FOH-PCOS, no.=8) and non-FOH (NFOH-PCOS, no.=7) according to the response of 17OH-progesterone to buserelin (a GnRH analogue) with respect to 15 controls. FOH-PCOS and NFOH-PCOS were compared for basal INSL3 levels. In addition, the effect of buserelin on INSL3 concentrations and the relationship between basal and buserelin-stimulated LH and 17OH-progesterone and INSL3 were evaluated. Results: Basal INSL3 levels were higher in FOH-PCOS than NFOH-PCOS (p=0.001) and controls (p=0.001), whereas they did not differ between NFOH-PCOS and controls. In addition, FOH-PCOS had a higher response of LH to buserelin with respect to NFOH-PCOS. Within all PCOS women the levels of INSL3 positively correlated with free testosterone (p=0.022) and negatively with SHBG (r= p=0.031). Moreover, positive correlations with the absolute increase of 17OH-progesterone (p<0.001) and with the LH area under the curve (p=0.001) after buserelin administration were found. In the multiple regression analysis INSL3 persisted significantly correlated only with 17OH-progesterone response to buserelin. Finally, INSL3 was not significantly modified after buserelin administration either in FOH-PCOS or in NFOH-PCOS. Conclusions: These data suggest that INSL3 is related to FOH in PCOS women, but this association seems not to be mediated by LH, further reinforcing the concept that a pathophysiological heterogeneity for ovarian hyperandrogenism in PCOS exists.