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.

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 NFOHPCOS 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 FOHPCOS 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.

Birth of a baby conceived from frozen oocytes of a 40-year-old woman.

The potential, limits and safety of oocyte freezing are still being explored. Female age may play a relevant role in treatment outcome. The present study is the first report of the birth and normal development of a baby conceived from frozen oocytes of a 40-year-old woman. IVF was carried out in an infertile 40-year-old woman, and seven metaphase II (MII) oocytes were obtained after ovarian stimulation. Three fresh oocytes were inseminated by intracytoplasmic sperm injection (ICSI), according to Italian law. Two embryos were transferred, but pregnancy did not occur. The four remaining MII oocytes were frozen (by slow freezing protocol) and ICSI was performed in the two oocytes surviving after thawing. Two embryos were obtained on day 2. Both embryos were transferred, resulting in a singleton pregnancy, and a healthy male baby was born. So far, the child (now 3 years old) has scored normally according to the WHO Child Growth Standards. The Denver Developmental Screening Test for psychomotor development was normal. This report demonstrates that conception and pregnancy from cryopreserved oocytes belonging to women up to 40 years of age is possible, and can yield normal children. This finding has implications for women who want to preserve their reproductive potential.

Increased clearance of cortisol by 5beta-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 5alpha-reduction of cortisol or impaired regeneration of cortisol by 11beta-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 microg 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 5alpha-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 microg/ml) and IR (2.4+/-1.1 microg/ml) than in NR (1.8+/-0.8 microg/ml) and controls (1.7+/-0.6 microg/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 (Delta(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 5beta-reduced cortisol metabolites (eg 5beta-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 5alpha-reduced cortisol metabolites or in 5alpha-dihydrotestosterone/testosterone ratio between groups. CONCLUSIONS: Adrenal androgen excess in PCOS is associated with increased inactivation of cortisol by 5beta-reductase that may lower cortisol blood levels and stimulate ACTH-dependent steroidogenesis.

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.

Modulation of folliculogenesis and steroidogenesis in women by graded menotrophin administration.

BACKGROUND: To test the effects of progressively decreasing dosages of exogenous LH we combined various amounts of HMG, containing FSH, LH and HCG, and highly purified (HP) FSH to treat 120 GnRH agonist-suppressed infertile female patients as candidates for controlled ovarian stimulation (COS). METHODS: Subjects were randomly assigned to four treatment groups that received the following daily i.m. gonadotrophin regimens: A, FSH 150 IU only; B, FSH 150 IU and LH activity 37.5 IU; C, FSH 150 IU and LH activity 75 IU; D, FSH 150 IU and LH activity 150 IU. FSH dose adjustments were allowed only after the 14th treatment day. Monitoring included transvaginal ultrasound at 2-day intervals and daily determinations of LH, FSH, estradiol (E(2)), progesterone, testosterone and HCG. RESULTS: Duration of COS was significantly shortened in patients receiving at least 75 IU daily of LH activity. Small (<10 mm diameter) pre-ovulatory ovarian follicle occurrence was inversely correlated with LH activity dose administered (r = -0.648, P < 0.0001) and serum HCG levels (r = -0.272, P < 0.01) but not to serum LH levels. Serum testosterone levels were positively correlated to the LH activity dose administered (r = 0.313, P < 0.001), while serum progesterone levels were positively correlated to the FSH dose administered (r = 0.447, P < 0.00001) but not to the LH activity dose administered. CONCLUSIONS: Firstly, HCG content considerably contributes to HMG activity; secondly, menotrophin LH activity content can reduce in a dose-dependent manner the occurrence of small pre-ovulatory follicles; and finally, contrary to common belief, enhanced FSH stimulation rather than LH activity appears to cause premature follicle luteinization during COS.

Stimulation and growth of antral ovarian follicles by selective LH activity administration in women.

Intensive FSH stimulation is a key tool of assisted reproduction technology but can cause severe complications through the development of an excessive number of small ovarian follicles. We tested the hypothesis that, in the late stages of ovulation induction, LH activity in the form of low-dose human CG (hCG) can stimulate and selectively modulate ovarian follicle function and growth, independently of FSH administration. Four groups of GnRH agonist-suppressed normoovulatory women (10 each group) received recombinant human FSH (r-hFSH) (150 IU/d) for 7 d followed by: group A, r-hFSH 150 IU/d alone; group B, r-hFSH 50 IU/d and hCG 50 IU/d; group C, r-hFSH 25 IU/d and hCG 100 IU/d; group D, hCG 200 IU/d alone. Despite several days of lowered or absent r-hFSH administration, 70% of hCG-treated patients successfully completed treatment. In these subjects, preovulatory E2 levels and large (>14 mm diameter) ovarian follicle development were not reduced; conversely, the number of small (<10 mm diameter) ovarian follicles was significantly decreased in groups B-D vs. group A. Low-dose hCG administration did not cause follicle luteinization. We conclude that, following FSH priming, LH activity administration can: 1) stimulate folliculogenesis for several days, in spite of rapidly declining FSH levels; and 2) hasten small follicle demise. Therefore, LH activity administration could be used to design radically novel ovulation induction regimens that, by partly or completely replacing mid-/late follicular phase FSH administration, may reduce costs and improve safety of assisted reproduction technology.

Luteinzing hormone activity in menotropins optimizes folliculogenesis and treatment in controlled ovarian stimulation.

Although the role that LH plays in folliculogenesis is still controversial, recent evidence points toward facilitatory actions of LH activity in ovulation induction. Thus, we compared the response to either highly purified FSH (75 IU FSH/ampoule; group A, 25 subjects) or human menopausal gonadotropin (75 IU FSH and 75 IU LH/ampoule; group B, 25 subjects) in normoovulatory GnRH agonist-suppressed women, candidates for intrauterine insemination. A fixed regimen of 2 daily ampoules of highly purified FSH or human menopausal gonadotropin was administered in the initial 14 days of treatment; menotropin dose adjustments were allowed thereafter. Treatment was monitored with daily blood samples for the measurement of LH, FSH, 17beta-estradiol (E(2)), progesterone, testosterone, hCG, inhibin A, and inhibin B, and transvaginal pelvic ultrasound was performed at 2-day intervals. Although preovulatory E(2) levels were similar, both the duration of treatment (16.1 +/- 0.8 vs. 12.6 +/- 0.5 days; P< 0.005) and the per cycle menotropin dose (33.6 +/- 2.4 vs. 23.6 +/- 1.1 ampoules; P < 0.005) were lower in group B. In the initial 14 treatment days the area under the curve of FSH, progesterone, testosterone, inhibin A, and inhibin B did not differ between the 2 groups, whereas LH, hCG, and E(2) areas under the curve were higher in group B. The occurrence of small follicles (<10 mm) and the inhibin B/A ratio in the late follicular phase were significantly reduced in group B. A nonsignificant trend toward a higher multiple gestation rate was present in group A (60% vs. 17%). We conclude that ovulation induction with LH activity-containing menotropins is associated with 1) shorter treatment duration, 2) lower menotropin consumption, and 3) reduced development of small ovarian follicles. These features can be exploited to develop regimens that optimize treatment outcome, lower costs, and reduce occurrence of complications such as multiple gestation and ovarian hyperstimulation.

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.

Luteinizing hormone activity supplementation enhances follicle-stimulating hormone efficacy and improves ovulation induction outcome.

Although FSH is essential to stimulate ovarian folliculogenesis, increasing physiological and clinical evidence suggests that moderate LH stimulation may also be critical for optimal follicle and oocyte development. Conversely, a clinical trend exists toward conducting controlled ovarian hyperstimulation (COH) in a LH-depleted environment, as recently developed gonadotropin preparations are devoid of LH activity, and endogenous LH is suppressed with GnRH analogs in most COH cycles. To investigate the role of LH activity during COH we supplemented highly purified (HP) FSH with low dose hCG in GnRH agonist-suppressed women. Twenty normoovulatory women were pretreated with a GnRH agonist and after 2 weeks were randomly assigned to receive HP FSH (150 IU/day) alone (group A; 10 patients) or combined with hCG (50 IU/day; group B; 10 patients). The HP FSH dose was increased after 14 days only in cases of inadequate response. Treatment was monitored with pelvic ultrasound and daily hormone determinations. None of the patients of group B and 8 of group A required more than 14 days of treatment and increments of the FSH dose. Folliculogenesis and 17beta-estradiol (E2) secretion progressed more rapidly and evenly in group B. Although preovulatory follicle number and E2 concentrations were comparable, patients in group B required a shorter stimulation time (12.5+/-0.6 vs. 17.3+/-0.7 days in group A; P < 0.0001) and a lower HP FSH dose (1725+/-84 vs. 2670+/-164 IU in group A; P < 0.0001). Serum levels of LH, E2, progesterone, and testosterone did not differ between the 2 groups; serum FSH was higher in group A. We conclude that LH activity promotes folliculogenesis in synergy with FSH in the mid- to late follicular phase and that low dose hCG coadministration optimizes COH by 1) enhancing FSH action, 2) accelerating ovarian follicle development, 3) shortening COH duration, 4) lowering HP FSH requirements, and 5) reducing COH cost. Thus, moderate LH activity in the follicular phase plays a positive physiological and clinical role in folliculogenesis and ovulation induction.

Subcutaneous administration of a depot gonadotropin-releasing hormone agonist induces profound reproductive axis suppression in women.

OBJECTIVE: To compare the i.m. and s.c. routes of depot GnRH agonist administration. DESIGN: Prospective, controlled pharmacokinetics study. SETTING: Volunteers in an academic research environment. PATIENT(S): Forty women with benign gynecologic disorders. INTERVENTION(S): Triptorelin administration (3.75 mg) at 28-day intervals for 6 consecutive months. Twenty patients were treated with IM triptorelin, and 20 patients were treated with SC triptorelin. MAIN OUTCOME MEASURE(S): Assessment of side effects, GnRH test results, and triptorelin, LH, FSH, estradiol, and progesterone levels. RESULT(S): The occurrence of injection site redness and itching and of some hypoestrogenic side effects was increased significantly in the SC group. Plasma triptorelin levels were significantly higher in the IM group in the first month of treatment; thereafter, the pattern reversed, with a nonsignificant trend toward higher plasma triptorelin levels in the SC group. Serum LH, FSH, estradiol, and progesterone levels were low after the first month of treatment and did not differ between the two treatment groups. On day 196 (2 months after the last depot triptorelin injection), triptorelin was still measurable and gonadotropins and gonadal steroids were still suppressed. Spontaneous menses returned significantly later in the SC group than in the IM group. CONCLUSION(S): Subcutaneous triptorelin can be administered by the patient. Both IM and SC triptorelin administration are cliniclly effective, but they result in different triptorelin pharmacokinetics. Subcutaneous triptorelin is associated with more prolonged amenorrhea than is IM triptorelin, suggesting enhanced pituitary-ovarian suppression. These results suggest that SC triptorelin may allow lower drug dosage administration and/or longer administration intervals.

Role of different GnRH agonist regimens in pituitary suppression and the outcome of controlled ovarian hyperstimulation.

Gonadotrophin-releasing hormone (GnRH) agonists have become irreplaceable addition to gonadotrophins in ovulation induction for assisted reproduction. Of the several schemes currently employed, long regimens appear to be maximally effective to optimize patient scheduling and to improve clinical results.

Is multiple pregnancy an unavoidable complication of ovulation induction? The case for pulsatile GnRH.

Ovarian hyperstimulation (OHS) and multiple pregnancy are dreaded complications of ovulation induction. The use of pulsatile GnRH permits to prevent the occurrence of OHS and results in few multiple pregnancies. Low-dose GnRH administration, avoidance of preovulatory hCG, patient selection, and the use of GnRH agonist pituitary desensitization in selected patients permits to limit multiple conceptions to a level comparable with the occurrence of this complication in normal unstimulated women.