Pulsatile gonadotropin-releasing hormone: clinical applications of a physiologic paradigm.

Gonadotropin-releasing hormone (GnRH) is a fundamental driver of human reproduction. A pulsatile pattern of GnRH secretion is essential to achieve pituitary stimulation, gonadotropin secretion, and normal gonadal function. Pulsatile GnRH administration is used to treat anovulation and male hypogonadotropic hypogonadism. Pulsatile GnRH ovulation induction is effective and safe because it allows to avoid ovarian hyperstimulation syndrome and reduce the occurrence of multiple pregnancies. This physiology-inspired therapeutic tool has also permitted to elucidate several pathophysiologic features of human reproductive disorders.

A randomized, controlled, first-in-patient trial of choriogonadotropin beta added to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol.

STUDY QUESTION: Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER: At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY: CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30-37 and 38-42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 µg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 620 women (30-42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE: The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0-2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was 43% per started cycle versus 28-39% in CG beta groups and 49% per transfer versus 38-50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6-52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0-10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION: The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS: The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER: 2017-003810-13 (EudraCT Number). TRIAL REGISTRATION DATE: 21 May 2018. DATE OF FIRST PATIENT'S ENROLMENT: 13 June 2018.

Are commercial warming kits interchangeable for vitrified human blastocysts? Further evidence for the adoption of a Universal Warming protocol.

PURPOSE: To study whether a new combination of different warming kits is clinically effective for vitrified human blastocysts. METHODS: This is a longitudinal cohort study analysing two hundred fifty-five blastocysts warming cycles performed between January and October 2018. Embryos were vitrified using only one brand of ready-to-use kits (Kitazato), whereas the warming procedure was performed with three of the most widely used vitrification/warming kits (Kitazato, Sage and Irvine) after patient stratification for oocyte source. The primary endpoint was survival rate, while the secondary endpoints were clinical pregnancy, live birth and miscarriage rates. RESULTS: We observed a comparable survival rate across all groups of 100% (47/47) in KK, 97.6% (49/50) in KS, 97.6% (41/42) in KI, 100% (38/38) in dKK, 100% (35/35) in dKS and 100% (43/43) in dKI. Clinical pregnancy rates were also comparable: 38.3% (18/47) in KK, 49% (24/49) in KS, 56.1% (23/ 41) in KI, 47.4% (18/38) in dKK, 31.4% (11/35) in dKS and 48.8% (21/ 43) in dKI. Finally, live birth rates were 29.8% (14/47) in KK, 36.7% (18/49) in KS, 46.3% (19/41) in KI, 36.8% (14/38) in dKK, 25.7% (9/35) in dKS and 41.9% (18/43) in dKI, showing no significant differences. CONCLUSION: This study confirmed the efficacy of applying a single warming protocol, despite what the "industry" has led us to believe, supporting the idea that it is time to proceed in the cryopreservation field and encouraging embryologists worldwide to come out and reveal that such a procedure is possible and safe.

Clinical roles and applications of progesterone in reproductive medicine: an overview.

The physiologic and clinical value of progesterone is undisputed and a cornerstone of human reproduction. Better understanding of the exact dynamics and effects of endogenous progesterone secretion, as well as its therapeutic actions, is critical to ensure optimal clinical results in artificial reproduction technology, and to enhance chances of successfully completing pregnancy. Novel progesterone-based drugs and administration regimens will provide clinicians with greater options to make the management and treatment of infertile couples less burdensome and more successful.

Rapid warming increases survival of slow-frozen sibling oocytes: a step towards a single warming procedure irrespective of the freezing protocol?

Nowadays, human oocytes/embryos are cryopreserved via slow freezing or vitrification. The aim of this study was to evaluate a rapid warming protocol for slow-frozen human oocytes based on the standard warming procedure for vitrification. This was a prospective study on 216 sibling oocytes randomized for either conventional rapid thawing or rapid warming with vitrification warming solution. The primary endpoint was morphological assessment of survival at 2h. Surviving oocytes were divided into two subgroups: (i) parthenogenetically activated; and (ii) fixed and observed for spindle/chromosome configuration. Secondary endpoints were parthenogenetic development and spindle/metaphase configuration. Survival rate with rapid warming was higher (92/102, 90.2%) than with rapid thawing (85/114, 74.6%; P=0.005), and after 3d of culture the rapidly warmed parthenotes had more blastomeres compared with those rapidly thawed (P=0.042). Meiotic spindle and chromosomal configuration were not significantly influenced by rapid warming or rapid thawing. The finding of this study allows IVF centres to increase the efficiency of oocyte slow freezing, enabling survival rates comparable to vitrification protocols, and potentially to optimize costs by using the same warming protocol for both slow-frozen and vitrified reproductive cells.

Sperm selection: effect on sperm DNA quality.

The selection of spermatozoa without DNA fragmentation and chromosomal diseases prior to assisted reproductive techniques helps to optimize the outcome of the treatment; in particular, sperm selection prior to in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is crucial. In fact, although ICSI has been successfully and safely applied worldwide for almost 20 years, at the present time we have no real knowledge regarding the hypothetical long-term side effects on ICSI adults, given the increased likelihood of spermatozoa with defective nuclear content fertilizing oocytes.In the case of DNA damage, the basal sperm DNA fragmentation rate can be significantly reduced by some sperm processing procedures that improve the percentage of spermatozoa with normal chromatin structure by filtering out DNA-damaged spermatozoa. After this first step, new advances in micromanipulation can be performed to choose the "ideal" mature spermatozoa for ICSI, reducing potential damage to the gametes. In fact, it is possible to prevent fertilization by DNA-damaged and chromosomal-unbalanced spermatozoa by selecting ICSI sperm by maturation markers such as hyaluronic acid or other zona pellucida receptors. Furthermore, novel noninvasive imaging techniques can be valid tools for helping in the morphological selection of ICSI spermatozoa.

Comparison of two ready-to-use systems designed for sperm-hyaluronic acid binding selection before intracytoplasmic sperm injection: PICSI vs. Sperm Slow: a prospective, randomized trial.

OBJECTIVE: To compare, in a strict, randomized way, the efficiency of two ready-to-use systems for hyaluronic acid (HA)-intracytoplasmic sperm injection (ICSI): an HA culture dish (PICSI Sperm Selection Device) and a viscous medium containing HA (Sperm Slow). DESIGN: Prospective, randomized study. SETTING: Medical center. PATIENT(S): Fifty subjects per treatment group (100 total). INTERVENTION(S): One hundred ICSI treatments were randomly carried out with PICSI or with Sperm Slow for sperm selection. Randomization was conducted with sealed envelopes. Intracytoplasmic sperm injection was performed by a single embryologist with 5 years' experience in HA-ICSI. PRIMARY OUTCOME MEASURE: good-quality embryo rate. SECONDARY OUTCOME MEASURES: oocyte fertilization, pregnancy and implantation rate, and the duration of the ICSI procedure. RESULT(S): The good-quality embryo rate was comparable between the two groups (58.5% with PICSI vs. 56% with Sperm Slow). Overall there were no statistically significant differences in secondary outcome measures except ICSI procedure duration, which was 3 minutes longer in the PICSI group. CONCLUSION(S): Both PICSI and Sperm Slow allow comparable clinical efficiency in selecting HA-bound spermatozoa. CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN72668039.

The pregnancy outcome in women with incidental diagnosis of septate uterus at first trimester scan.

BACKGROUND: Septate uterus seems to be strongly associated with an adverse pregnancy outcome. However, the possible relationship between septate uterus and miscarriage has only been retrospectively ascertained. The aim of our study was to describe the reproductive outcome in women with incidental diagnosis of malformed uterus at first trimester scan. METHODS: Women at their first pregnancy attending our centre for a routine viability scan with an incidental suspicion of uterine anomaly at standard sonography were submitted to transvaginal volume ultrasound. All the cases with a 3D diagnosis of septate uterus were prospectively recruited and followed up. RESULTS: Overall 24 patients with a single intrauterine pregnancy were included at a median gestational age of 8.2 weeks. The cumulative pregnancy progression rate, as quoted by Kaplan-Meier algorithm, was 33.3% due to the occurrence of early (≤ 13 weeks) or late miscarriages (14-22 weeks) in 13 and 3 cases, respectively. CONCLUSION: The pregnancy outcome is poor if a septate uterus is incidentally diagnosed in the early stage of a viable intrauterine pregnancy.

A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2).

OBJECTIVE: To report a washing procedure, to be performed as frozen specimens are taken out of cryobanks, to minimize the risk of hypothetical culture contamination during thawing. DESIGN: Basic research. SETTING: Private assisted reproduction center. INTERVENTION(S): Two batches of liquid nitrogen (LN(2)) were experimentally contaminated, one with bacteria (Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia) and the other with fungi (Aspergillus niger). Two hundred thirty-two of the most common human gamete/embryo vitrification carriers (Cryotop, Cryoleaf, Cryopette) were immersed in the contaminated LN(2) (117 in the bacteria and 25 in the fungi-contaminated LN(2)). The carriers were tested microbiologically, one group without washing (control) and the other after three subsequent washings in certified ultraviolet sterile liquid nitrogen (SLN(2)). The carriers were randomly allocated to the "three-wash procedure" (three-wash group, 142 carriers) or "no-wash" (control group, 90 carriers) using a specific software tool. MEAN OUTCOME MEASURE(S): Assessment of microorganism growth. RESULT(S): In the no-wash control group, 78.6% of the carriers were contaminated by the bacteria and 100% by the fungi. No carriers were found to be contaminated, either by bacteria or fungi, after the three-wash procedure. CONCLUSION(S): The three-wash procedure with SLN(2) produced an efficient decontamination of carriers in extreme experimental conditions. For this reason, this procedure could be routinely performed in IVF laboratories for safe thawing of human specimens that are cryostored in nonhermetical cryocontainers, particularly in the case of open or single-straw closed vitrification systems.

Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos.

OBJECTIVE: To evaluate the efficacy of ultraviolet (UV) irradiation for rapid microbial decontamination of liquid nitrogen (LN2). DESIGN: Basic research. SETTING: Private assisted reproduction center. ANIMAL(S): Microorganisms (bacteria and fungi). INTERVENTION(S): Two stainless steel open dewars containing LN2 were contaminated in a two-step experiment with high titers of cultures of bacteria (Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Escherichia coli) and fungi (Aspergillus niger). One of the two dewars was subsequently exposed to UV irradiation at 253.7 nm to obtain a rapid microbial decontamination before the complete evaporation of LN2. MAIN OUTCOME MEASURE(S): Detection of the micro-organisms in LN2 after UV sterilization through the assessment of bacterial and fungal growth in minimal and selective Petri dishes. RESULT(S): None of the contaminating micro-organisms were detected in LN2 after UV sterilization. CONCLUSION(S): Decontamination of LN2 with UV irradiation is feasible and straightforward. The fact that LN2 can be quickly and safely sterilized should encourage the wider application of human oocyte and embryo vitrification with "open carriers."

Efficiency of hyaluronic acid (HA) sperm selection.

PURPOSE: Hyaluronic 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. METHODS: We 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. RESULTS: This study showed that Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation. CONCLUSIONS: If 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.

"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).