Microgravity effects on frozen human sperm samples.

PURPOSE: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (µg) exposure on human frozen sperm samples. METHODS: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 s of microgravity for each parabola. RESULTS: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after µg exposure. Comparing the study group (µg) and the control group (1 g), similar results were obtained in the main parameters studied: sperm motility (M/ml) 13.72 ± 12.57 vs 13.03 ± 12.13 (- 0.69 95% CI [- 2.9; 1.52]), progressive a + b sperm motility (%) 21.83 ± 11.69 vs 22.54 ± 12.83 (0.03 95% CI [- 0.08; 0.15]), sperm vitality (%) 46.42 ± 10.81 vs 44.62 ± 9.34 (- 0.04 95% CI [- 0.13; 0.05]), morphologically normal spermatozoa (%) 7.03 ± 2.61 vs 8.09 ± 3.61 (0.12 95% CI [0.01; 0.24]), DNA sperm fragmentation by SCD (%) 13.33 ± 5.12 vs 13.88 ± 6.14 (0.03 95% CI [- 0.09; 0.16]), and apoptotic spermatozoa by MACS (%) 15.47 ± 15.04 vs 23.80 ± 23.63 (- 0.20 95% CI [- 0.66; 1.05]). CONCLUSION: The lack of differences obtained between frozen samples exposed to µg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT03760783.

Clinically significant intra-day variability of serum progesterone levels during the final day of oocyte maturation: a prospective study with repeated measurements.

STUDY QUESTION: Is there significant variability in progesterone levels during the final day of oocyte maturation in women undergoing ovarian stimulation? SUMMARY ANSWER: Progesterone levels drop from the basal level up to 44% during the final day of oocyte maturation in women undergoing ovarian stimulation. WHAT IS KNOWN ALREADY: It has been suggested that elevated progesterone levels on the final day of ovarian stimulation may be related to poorer outcomes in in vitro fertilization fresh cycles due to a negative impact on the endometrium. However, despite conflicting results regarding the actual effect of progesterone on pregnancy rates and the lack of a well-established cut off, currently many IVF patients have their embryo transfer deferred when progesterone values surpass a threshold of 1.5 ng/ml on the day of ovulation triggering. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study conducted in 22 oocyte donors of a university-affiliated fertility centre between November 2017 and January 2018. We calculated the sample size to detect a difference of 15% between the first and last progesterone measurements with a 5% false-positive rate in a two-sided test with 80% statistical power and a 95% confidence interval (CI). PARTICIPANTS/MATERIALS, SETTING, METHODS: Progesterone circulating levels were evaluated at four different times during the final day of oocyte maturation (08:00, 12:00, 16:00 and 20:00) before ovulation triggering in healthy oocyte donors. A flexible antagonist protocol was used, and ovarian stimulation was achieved with recombinant follicle-stimulating hormone (FSH) in all cases. The pairwise percentage differences in progesterone levels for each patient were calculated. Univariate linear regression analysis was adopted in order to evaluate variables associated with progesterone levels on the first measurement. The intra-day variability of progesterone was analysed using mixed models. MAIN RESULTS AND THE ROLE OF CHANCE: Mean serum progesterone values at 08:00, 12:00, 16:00 and 20:00 were 1.75 ng/ml, 1.40 ng/ml, 1.06 ng/ml and 0.97 ng/ml. The progesterone difference between 08:00 and 20:00 was 0.77 (95% CI, 0.56-0.99), which is equivalent to a 44% decline in the mean progesterone values between the first (08:00) and the last determination (20:00; P < 0.001). Among those patients with basal (08:00) progesterone levels >1.5 ng/ml (n = 10), 70% (n = 7) showed levels reduced to <1.5 ng/ml on the last determination of the day (20:00). A mixed model analysis revealed that the progesterone reduction during the day was significantly associated with time and total recombinant FSH dose administered. LIMITATIONS, REASONS FOR CAUTION: Only young healthy oocyte donors stimulated with an antagonist protocol using recombinant FSH were included. Extrapolation to the general IVF population, with different stimulation protocols and gonadotropins, needs to be confirmed. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that a single progesterone determination on the final day of oocyte maturation is not reliable enough to make clinical decisions due to the enormous variation in progesterone during the day. Further studies are needed to better define the impact of the follicular progesterone rise on the endometrium of IVF cycles. STUDY FUNDING/COMPETING INTEREST(S): Funding was granted from Fundació Santiago Dexeus Font. N.P.P. received unrestricted grants and/or lectures fees from Roche Diagnostics, MSD, Merck, Ferring Pharmaceuticals, IBSA, Theramex and BESINS International, not associated with the current study. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov NCT03366025.

Low serum progesterone the day prior to frozen embryo transfer of euploid embryos is associated with significant reduction in live birth rates.

A retrospective cohort study was performed to examine whether, in artificial endometrial preparation for frozen embryo transfer (FET) cycles, progesterone (P) levels the day prior to embryo transfer of euploid embryos have an impact on pregnancy outcomes. In a private university clinic, 244 FETs between January 2016 and June 2017 were analyzed. Endometrial preparation was achieved with estradiol valerate and vaginal micronized progesterone. Serum P and estradiol levels the day prior to embryo transfer were measured. A multivariable analysis to assess the relationship between serum P level and pregnancy outcomes was performed, adjusted for confounding variables. Mean P value was 11.3 ± 5.1 ng/ml. Progesterone levels were split in quartiles: Q1: ≤ 8.06 ng/ml; Q2: 8.07-10.64 ng/ml; Q3: 10.65-13.13 ng/ml; Q4: > 13.13 ng/ml. Patients included in the lower P quartile had a significantly higher miscarriage rate and significantly lower live birth rate (LBR) compared to the higher ones. A low serum P level (≤ 10.64 ng/ml) one day before FET is associated with a lower pregnancy and LBR following FET of euploid embryos.

How does vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes.

STUDY QUESTION: How does vitrification affect oocyte viability? SUMMARY ANSWER: Vitrification does not affect oocyte viability in oocyte donation cycles. WHAT IS KNOWN ALREADY: Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. STUDY DESIGN, SIZE, DURATION: This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. MAIN RESULTS AND ROLE OF CHANCE: A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate (40.8 versus 33.3%) or implantation rate (21.8 versus 26.8%). LIMITATIONS, REASONS FOR CAUTION: The oocytes were donated by healthy, young women (≤35 years) and these results cannot be extrapolated to other populations. WIDER IMPLICATIONS OF THE FINDINGS: Outcomes obtained with vitrified oocytes are as good as with fresh oocytes and the use of vitrification can be extended to new applications, e.g. accumulation of oocytes from successive stimulations for preimplantation genetic diagnosis, for patients at risk of ovarian hyperstimulation syndrome or in patients needing to preserve their fertility. STUDY FUNDING/COMPETING INTEREST(S): This work was done under the auspices of the Càtedra d'Investigació en Obstetrícia i Ginecologia of the Universitat Autònoma de Barcelona.

Assisted reproductive technology and ovarian cancer.

Ovarian cancer has the highest mortality among all gynaecological cancers, being multiparity and oral contraceptive use the most important protective factors. According to both the "incessant ovulation" and "increased gonadotrophin" theories, fertility drugs might have an association with the development of ovarian cancer, as has been reported by some studies. However, infertility and nulliparity may act as confounding factors and most studies regarding this issue are hampered by methodological limitations. It seems that female infertility may be associated with a modest increase in ovarian cancer risk in those patients who remain nulligravid despite long periods of unprotected intercourse. Globally, most studies are reassuring in not showing a link between the use of fertility drugs and an increased risk of ovarian cancer. Nonetheless, further research in well-designed studies is warranted.

Couples' opinions regarding the fate of surplus frozen embryos.

With the passing of Act 45/2003, research with viable human embryos became legal in Spain. Since then, Institut Universitari Dexeus has been in contact with couples whose embryos had been frozen for more than 2 years to inform them about the new legal options and gather their opinions. A reply was received from 35.9% of the couples contacted, with the following results: 33.3% wished to preserve the embryos for their own use, 30.0% wished to donate the embryos for embryonic stem cell research, 20.2% wished to donate the embryos to third parties for reproductive purposes and 10.3% wished to terminate the cryopreservation process without further use. The couples who chose to donate the embryos for research were asked to give written informed consent to the donation of their embryos for a specific project. The possibility of donating embryos for research has been well received by the couples, and offers a solution to those who wish to make neither a further attempt for pregnancy nor a donation with reproductive goals. Donation for research purposes is considered a preferable alternative to disposal.

Generation of cardiomyocytes from new human embryonic stem cell lines derived from poor-quality blastocysts.

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently, hES cell lines are derived from surplus embryos from assisted reproduction cycles, independent of their quality or morphology. Here, we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore, we show that the self-renewal, pluripotency, and differentiation ability of hES cell lines derived from either source are comparable. Finally, we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine.

Birth after transfer of frozen-thawed vitrified biopsied blastocysts.

PURPOSE: To present a case describing the birth of a healthy female after the replacement of vitrified biopsied embryos after Preimplantation Genetic Diagnosis. METHOD: A descriptive case report of a single patient. RESULTS: Our patient carrier of an X-linked disease became pregnant and as a result a healthy girl was born. CONCLUSIONS: This report shows that blastocysts obtained from biopsied embryos can be successfully cryopreserved by a simple, secure and low-cost vitrification method using a Hemi-straw support.

Prevention of multiple pregnancy following IVF in Spain.

Since the development of assisted reproduction techniques most countries have witnessed increased rates of multiple pregnancy. Despite the guidelines proposed by various scientific societies these rates continue to be abnormally high. In Spain, as in other Mediterranean countries, a greater number of embryos are transferred than in northern and central European countries and the incidence of multiple pregnancies is greater in comparison. Effective strategies must be established to prevent multiple pregnancy without reducing overall pregnancy rates. In the authors' institute, taking into account the authors' experience, the relevant literature, and despite the limitation of retrospective studies, it is recommended that a maximum of two embryos are transferred in young women with good quality embryos at the time of transfer. The transfer of three embryos is only recommended in women >or=38 years who have one or no good quality embryos available at the time of transfer. The responsibility for preventing multiple pregnancy lies with health professionals, who must be aware of the risks involved in twin and triplet pregnancy. Couples must be provided with objective information before starting an IVF cycle. Professional societies should highlight the problem and make suitable recommendations.

A prospective trial comparing oocyte donor ovarian response and recipient pregnancy rates between suppression with gonadotrophin-releasing hormone agonist (GnRHa) alone and dual suppression with a contraceptive vaginal ring and GnRH.

BACKGROUND: Contraceptive treatment before gonadotrophin-releasing hormone agonist administration presents advantages in women with a tendency to hyper-response and simplifies donor-recipient treatment synchronization. This study compares response to gonadotrophin stimulation under hypophyseal suppression in oocyte donors with or without vaginal contraceptive pretreatment. METHODS: One hundred and ninety oocyte donors were recruited in a single centre and prospectively assigned to one of two treatment groups, according to the day of the week menstruation initiated: Group VC-, no prior vaginal contraceptive and Group VC+, prior vaginal contraceptive. RESULTS: VC+ patients presented a significantly higher cancellation rate, lower plasma estradiol levels and fewer follicles >12 mm on the day of hCG, versus the VC- group. Number of oocytes recovered was significantly lower in the VC+ group. All the cases of severe ovarian hyperstimulation syndrome (SOHSS) were in the VC- group. Pregnancy rates by embryo transfer to synchronic recipients were similar between VC+ and VC- (59.5 versus 57.9%, respectively). CONCLUSIONS: Vaginal contraceptive pretreatment resulted in a higher ovarian suppression, whereas SOHSS rate was lower than in donors who did not receive pretreatment. There were no differences in pregnancy rates between the two groups of synchronic oocyte recipients.

Preimplantation genetic diagnosis in patients with male meiotic abnormalities.

Indications and candidates for preimplantation genetic diagnosis (PGD) have increased in recent years. This study evaluates whether IVF-intracytoplasmic sperm injection (ICSI) results could be improved by selecting embryos through PGD-AS (aneuploidy screening) in couples in whom the male partner presents meiotic abnormalities. Two hundred and fifty-six embryos were biopsied and 183 were suitable for analysis (73.2%). Ninety-two embryos showed normal chromosomal analysis (50.3% of the analysed embryos and 57.5% of the diagnosed embryos). Pregnancy, abortion and implantation rates were compared with 66 IVF-ICSI cycles performed in 44 patients with meiotic abnormalities without PGD (control group). No statistically significant differences in the pregnancy rate (52 versus 43.9%), implantation rate (32.1 versus 23.5%) and miscarriage rate (15.4 versus 10.3%) were observed between the groups. Although the embryos obtained from men with meiotic abnormalities showed a high frequency of chromosome abnormalities, no improvements in pregnancy and implantation rates were obtained after PGD-AS in the series analysed.

Applications of ovarian tissue transplantation in experimental biology and medicine.

Nowadays, high-dose chemotherapy and radiotherapy treatments for cancer are more effective but can severely affect the ovarian follicular store, compromising the fertility of surviving young patients. A promising alternative to prevent fertility loss in these patients is the cryopreservation and transplantation of ovarian tissue. Slices of animal and human ovarian tissue have been shown to survive the cryopreservation process. After transplantation, follicular development and restoration of hormone secretion have been observed in animal and human studies. This review addresses recent developments on ovarian tissue transplantation in animals and humans. We also illustrate the indications and technical difficulties of the procedure and the ethical issues that should be considered.

Preimplantation genetic diagnosis: patients' experiences and attitudes.

BACKGROUND: This study aims to report the experiences and attitudes of patients who have undergone preimplantation genetic diagnosis (PGD). The extent to which this technique is acceptable to the individuals for whom it is intended is relatively unexplored, and remains a crucial issue that may ultimately determine the value of PGD as an alternative to prenatal diagnosis in high-risk couples. METHODS: An information sheet and questionnaire was distributed to 67 couples who had been treated at the Hammersmith Hospital, London and the Dexeus Institute, Barcelona. RESULTS: One-third of patients had an affected child, over half had previous experience of conventional prenatal diagnosis and over one-third had had terminations of pregnancy because of a genetic risk. Patients perceive the main advantage of PGD to be that only unaffected embryos are transferred to the uterus and thus therapeutic termination of pregnancy can be avoided; the main disadvantage is the low success rate. A total of 41% of patients found the treatment cycle extremely stressful, and, of the 20 patients who had experienced both prenatal diagnosis and PGD, 40% of patients found PGD less stressful, although 35% experienced more stress. Of those couples who contemplated a further pregnancy 76% would choose PGD, 16% would opt for prenatal diagnosis, and 8% no tests at all. CONCLUSIONS: The experience of prenatal diagnosis and termination of pregnancy can be an unwelcome memory and this leads to a demand for an alternative approach. Our data suggest that PGD is acceptable to patients and is a valuable alternative to prenatal diagnosis.

Women with poor response to IVF have lowered circulating gonadotrophin surge-attenuating factor (GnSAF) bioactivity during spontaneous and stimulated cycles.

BACKGROUND: Up to 13% of IVF cancellations are due to poor responses during down-regulated cycles. Because premature luteinization occurs more frequently in older or "poor responder" patients, defective production of gonadotrophin surge-attenuating factor (GnSAF) may be involved. METHODS: Nine women with normal previous IVF response (NORM) and 9 with previous poor IVF response (POOR) were monitored in a spontaneous cycle (blood samples: days 2, 7, 11, 15 and 20) and then stimulated with recombinant human FSH (rFSH) under GnRH agonist (blood samples: treatment days GnRH agonist + 2, GnRH agonist + 7, day of HCG administration and days HCG + 1 and HCG + 8). LH, FSH, estradiol, progesterone and inhibin-A and -B were assayed in individual samples while GnSAF bioactivity was determined in samples pooled according to day, cycle and IVF response. RESULTS: During spontaneous cycles LH, steroids and inhibins were similar between NORM and POOR women, FSH was elevated in POOR women (4.9 +/- 0.3 versus 6.7 +/- 0.6 mIU/l, P < 0.01) and GnSAF bioactivity was detectable on days 2, 7 and 11 in NORM women only. During IVF cycles inhibin-A and -B rose more markedly in NORM than POOR women. Similarly GnSAF production peaked on day GnRH agonist + 7 in NORM women, but on the day of HCG administration in POOR women. CONCLUSIONS: Defects in ovarian responsiveness to FSH include reduced GnSAF production. This suggests that GnSAF should be investigated as a marker of ovarian reserve once an immunoassay becomes available.

Risk factors for high-order multiple implantation after ovarian stimulation with gonadotrophins: evidence from a large series of 1878 consecutive pregnancies in a single centre.

BACKGROUND: High-order multiple pregnancies (triplets or more) have a large adverse impact on perinatal morbidity and mortality as well as important economic consequences. Most triplets and higher births are due to ovulation induction alone or in combination with intrauterine insemination (IUI) rather than to in-vitro fertilization (IVF). The present investigation was undertaken to determine whether there were specific variables that related to patient clinical characteristics (age of the woman, duration of infertility, type of infertility, body mass index, basal FSH and LH concentrations), treatment characteristics (initial dose of gonadotrophins, total dose of gonadotrophins administered, number of days of ovarian stimulation, insemination procedure, number of spermatozoa inseminated in patients undergoing IUI, type of luteal support), and ovarian response (oestradiol serum concentrations, number and size of follicles) that might be associated with the occurrence of high-order multiple implantation in order to develop a prediction model. METHODS: This study employed univariate, multivariate and receiver-operating characteristic (ROC) analysis of a large series of 1878 consecutive pregnancies obtained in cycles stimulated with gonadotrophins. Of them, 1771 (94.3%) were low-order pregnancies (1477 singletons and 294 pairs of twins) and 107 (5.7%) were high-order pregnancies. RESULTS: Predictive variables in the multivariate analysis were age of the woman, serum oestradiol concentrations and number of follicles >10 mm on the day of HCG injection. Stratification of the number of follicles into three categories (1 to 3, 4 to 5, and >5 follicles respectively), peak serum oestradiol and woman's age according to the ROC curves, showed that the risk of high-order multiple implantation correlated significantly with increasing total number of follicles and was significantly increased in women with a serum oestradiol >862 pg/ml and aged < or =32 years. CONCLUSIONS: This three-variable model can help to identify patients at high-risk for high-order multiple pregnancy in ovulation induction cycles.

Ultrasound-guided embryo transfer: immediate withdrawal of the catheter versus a 30 second wait.

It is essential to deposit embryos as gently as possible during IVF, avoiding manoeuvres that might trigger uterine contractions which could adversely affect the results of this treatment. The time during which the embryo transfer catheter remains in the cervical canal might be related to stimulation of contractions. This study investigates the influence that the time interval before withdrawal of the catheter after ultrasound (US)-guided embryo deposit might have on the pregnancy rate in patients under IVF cycles. A total of 100 women about to undergo transfer of at least two optimal embryos was studied. The women were prospectively randomized into two groups: (i) slow withdrawal of the catheter immediately after embryo deposit (n = 51); and (ii) a 30 s delay before catheter withdrawal (n = 49). The pregnancy rates for transfer in the two groups were 60.8 and 69.4% respectively, with no significant differences. There were no statistically significant differences in pregnancy rates between the two patient groups. The results indicate either that the waiting interval was insufficient to detect differences, or that the retention time before withdrawing the catheter is not a factor that influences pregnancy rate.

Reflections on the cost-effectiveness of recombinant FSH in assisted reproduction. The clinician's perspective.

PURPOSE: To analyze the relative cost-effectiveness of recombinant FSH (rFSH) and urinary FSH (uFSH) in assisted reproduction techniques (ART). METHODS: Calculation of the average cost-effectiveness ratio and the incremental cost-effectiveness ratio to compare costs and effects (pregnancy rates) of the two therapeutic options (rFSH and uFSH). RESULTS: Assuming that the cost of the procedure per ART cycle is between 100,000 pesetas (601 euro) and 150,000 pesetas (901.52 euro), and pricing the GnRH analogues used for pituitary suppression at 35,000 pesetas (210.3 euro), the cost-effectiveness ratio is better for rFSH than for uFSH, implying that the cost per pregnancy is lower when the recombinant preparation is used. CONCLUSIONS: In ART, the use of rFSH is more cost-effective than uFSH.

Cervical ectopic twin pregnancy: diagnosis and conservative treatment: case report.

A case of cervical ectopic twin pregnancy with cardiac activity in both embryos is presented. It was diagnosed in the eighth week of gestation by ultrasonography, and treated conservatively with intra-amniotic administration of methotrexate under ultrasonographic guidance followed by curettage. This procedure allows subsequent gestations.