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.

Stimulation protocols for poor responders and aged women.

There is a general consensus on the clinical fact that the more embryos replaced the higher pregnancy rates are achieved. For this reason those IVF cycles with a low response and a reduced number of oocytes and embryos will have very few chances of producing a pregnancy. It is very important to diagnose, by means of the anamnesis and hormonal tests which patients are most likely to present a poor response to conventional ovarian stimulation protocols. It is mandatory to know the patient's plasmatic levels of FSH and estradiol together with personal data such as the age and the previous history of the patient. Only young poor responders with a normal basal hormonal profile will have some chances that by applying new protocols and combining new drugs, improve their response and have higher pregnancy rates. For the old poor responders who have already failed to alternative protocols including natural cycles, oocyte donation is the last and best hope.

Two years of assisted fertilization by partial zona dissection in male factor infertility patients.

OBJECTIVE: To assess partial zona dissection in our routine IVF-ET program over a 2-year period. DESIGN: Partial zona dissection before insemination on the day of oocyte collection or 24 hours after unsuccessful conventional IVF. In a subgroup of patients, oocytes were randomized to either partial zona dissection before insemination or IVF. SETTING: University infertility clinic. PATIENTS: Couples who suffered principally from male factor infertility or who had failed fertilization previously. INTERVENTIONS: Micromanipulation of oocytes with partial zona dissection. MAIN OUTCOME MEASURES: Comparison of fertilization rate, embryo morphology, and implantation rate between partial zona dissection inseminated oocytes and conventional IVF inseminated oocytes (controls). RESULTS: Five pregnancies were established in 199 patients. The incidence of polyspermy was significantly higher in the partial zona dissection group than in conventional IVF (4.8% versus 1.3%). There were no significant differences in the remaining parameters. The fertilization rate of partial zona dissection and reinsemination was significantly higher than conventional IVF insemination (13.6% versus 4.5%) but similar to the rate obtained when partial zona dissection was applied before insemination (13.6% versus 15.7%). CONCLUSIONS: Oocytes treated by partial zona dissection did not exhibit a greater fertilization rate than conventional IVF inseminated oocytes. Partial zona dissection may not be a useful technique for treating severe male factor infertility.

Spermatozoa selection in discontinuous Percoll gradients for use in artificial insemination.

In a preliminary study of the ejaculate of 93 men, changes in motility and hypo-osmotic swelling before and after sperm separation by discontinuous Percoll gradients were evaluated. Both parameters improved significantly (P less than 0.01) in Percoll-separated spermatozoa. In a second stage, 99 couples underwent intrauterine insemination of separated semen by Percoll gradients. The population had infertility for a mean of 5.6 years. In a 1-year period, a total of 344 intrauterine insemination cycles were completed. Failure was considered when conception did not occur within four treatment cycles. The mean number of cycles per successful couple before pregnancy was 1.52. An overall 21% pregnancy rate was achieved (16% for oligoasthenospermia and 24% for asthenospermia). Sperm separation in Percoll gradients is a useful technique for intrauterine insemination in some cases of male subfertility.

Evaluation of cytogenetic analysis for clinical preimplantation diagnosis.

OBJECTIVE: To evaluate the feasibility of using cytogenetic analysis in preimplantation diagnosis. DESIGN: Two different biopsy protocols (chemical drilling and zona cutting) and two fixation methods were tested in a mouse model. Afterwards, the efficiency of obtaining chromosome preparations from untransferable human embryos depending on the method used to obtain the blastomeres (embryos biopsy or removal of the zona pellucida and blastomere disaggregation) was determined. The chances of obtaining chromosome preparations depending on the type of embryo (haploid, diploid, triploid, and apparently unfertilized) were also evaluated. RESULTS: Results from the mouse model showed that chemical drilling yields better results than cutting in terms of metaphases per biopsied embryo and surviving rate after biopsy. In human embryos, biopsy of diploid embryos produced 46.6% chromosome preparations, while 29% were obtained after blastomere disaggregation and 20.4% when biopsying triploid embryos. CONCLUSIONS: These results suggest that the disaggregating procedure and triploid embryos cannot be considered as good models to assess the feasibility of cytogenetic analysis in preimplantation diagnosis. Poor chromosome quality and loss during fixation are the main problems to use cytogenetics in preimplantation diagnosis; a combination of cytogenetics and other techniques is suggested in cases of balanced translocations.

Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program.

OBJECTIVE: To evaluate the frequency of disomy (for chromosomes X, Y, and 18) and of diploidy in the spermatozoa of infertile men undergoing intracytoplasmic sperm injection (ICSI). DESIGN: Prospective analysis of sperm nuclei by fluorescence in situ hybridization (FISH). SETTING: University-affiliated IVF-ICSI program. PATIENT(S): Semen samples from 19 patients participating in an IVF-ICSI program. INTERVENTION(S): Semen samples were analyzed and prepared for FISH. MAIN OUTCOME MEASURE(S): Semen parameters were evaluated. The frequency of disomy for chromosomes X, Y, and 18 and the frequency of diploidy were analyzed by FISH. RESULT(S): A total of 9,373 spermatozoa from 19 infertile patients were analyzed and compared with spermatozoa from a control group of 5 healthy men. No differences in the frequency of disomy 18 were found, but statistically significant differences in the incidence of sex chromosome disomy and of diploidy were observed. CONCLUSION(S): The study of sperm nuclei by FISH is useful to improve genetic counseling in infertile patients selected for ICSI.

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.

Treatment of ectopic pregnancy: the new gynaecological dilemma.

Recent diagnostic advances have resulted in earlier and more consistent diagnosis of ectopic pregnancy. This may be responsible, in part, for the increasing incidence of the condition. Also, early diagnosis of ectopic pregnancies has prompted conservative surgical treatment, the use of medical therapy and even expectant management in selected cases. The present article analyses the following main controversial questions concerning the management of ectopic pregnancy: whether to treat or not, and how to apply expectant management; whether therapy should be conservative or radical; whether conservative management should be surgical or medical; whether surgical intervention should be laparotomic or laparoscopic; and whether medical therapy should be systemic or local.

Hyperprolactinemia: an inductor of neoplastic changes in endometrium? A report of two cases.

Estrogen-dependent endometrial carcinomas habitually arise through a precursor lesion such as endometrial hyperplasia. The hormonal environment of these patients remains unclear. Two cases of neoplastic changes in the endometrium of patients with a chronic hyperprolactinemia are presented. Despite the treatment with bromocriptine both patients developed endometrial adenocarcinomas. Possible mechanisms involved in the pathogenesis of these tumors are discussed.

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.