Optimisation of the timing of fertilisation assessment for oocytes cultured in standard incubation: lessons learnt from time-lapse imaging of 78 348 embryos.

STUDY QUESTION: Using time-lapse data, can the current consensus for the timing of fertilisation assessment of oocytes, cultured in standard incubation, be optimised? SUMMARY ANSWER: The optimum time to perform fertilisation assessment for oocytes cultured in standard incubation is 16.5 ± 0.5 h post-insemination (hpi), and the current consensus requires modification in order to minimise the chance of fertilisation being missed. WHAT IS KNOWN ALREADY: Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may otherwise be missed in standard incubation. According to expert consensus, the optimum time to perform the assessment of fertilisation is 17 ± 1 hpi. STUDY DESIGN, SIZE, DURATION: A retrospective, multicentre analysis utilised data obtained from 54 746 ICSI-derived embryos and 23 602 IVF-derived embryos cultured in time-lapse incubation between January 2011 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using time-lapse imaging (TLI), the precise time of pronuclei appearance and disappearance was recorded, where applicable, and the number of oocytes with two pronuclei observable during 10 30-min intervals from 15 hpi to 20 hpi was determined. MAIN RESULTS AND THE ROLE OF CHANCE: Between 15 and 17.5 hpi, the average number of oocytes exhibiting normal fertilisation, elicited as two pronuclei, was 98.19% with the highest proportion of oocytes having visible pronuclei at 16-16.5 hpi (98.32%). At 18-18.5 hpi, the number of visible pronuclei reduced to 95.53% and continued to fall to 87.02% at 19.5-20 hpi. LIMITATIONS, REASONS FOR CAUTION: The authors' expectation is that these findings are transferable to other settings, however it is possible that, with alternative culture media and incubation environments, calibration of this timing may be required. As data cannot readily be recorded for pronuclear appearance for IVF-derived embryos, it is not possible to determine the optimum time to perform the fertilisation assessment for IVF-derived embryos. WIDER IMPLICATIONS OF THE FINDINGS: By fine-tuning the time at which fertilisation assessment takes place the accuracy of the assessment can be increased to maximise the number of fertilised oocytes identified, thereby increasing the number of usable embryos for the patient. Without TLI and following current consensus guidelines, over 11% (n = 3000) of oocytes would have been marked as unfertilised within this cohort. Further to this, depending on the time of a standard fertilisation assessment, up to 300 embryos which resulted in live births could have been categorised as unfertilised, as they presented no visible pronuclei at the conventional assessment time-point. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a minor shareholder in CARE Fertility Limited. Validated algorithmic time-lapse embryo selection is offered to patients at CARE Fertility at an additional charge as an adjuvant treatment option. TRIAL REGISTRATION NUMBER: N/A.

Predicting the chance of live birth for women undergoing IVF: a novel pretreatment counselling tool.

STUDY QUESTION: Which pretreatment patient variables have an effect on live birth rates following assisted conception? SUMMARY ANSWER: The predictors in the final multivariate logistic regression model found to be significantly associated with reduced chances of IVF/ICSI success were increasing age (particularly above 36 years), tubal factor infertility, unexplained infertility and Asian or Black ethnicity. WHAT IS KNOWN ALREADY: The two most widely recognized prediction models for live birth following IVF were developed on data from 1991 to 2007; pre-dating significant changes in clinical practice. These existing IVF outcome prediction models do not incorporate key pretreatment predictors, such as BMI, ethnicity and ovarian reserve, which are readily available now. STUDY DESIGN, SIZE, DURATION: In this cohort study a model to predict live birth was derived using data collected from 9915 women who underwent IVF/ICSI treatment at any CARE (Centres for Assisted Reproduction) clinic from 2008 to 2012. Model validation was performed on data collected from 2723 women who underwent treatment in 2013. The primary outcome for the model was live birth, which was defined as any birth event in which at least one baby was born alive and survived for more than 1 month. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were collected from 12 fertility clinics within the CARE consortium in the UK. Multivariable logistic regression was used to develop the model. Discriminatory ability was assessed using the area under receiver operating characteristic (AUROC) curve, and calibration was assessed using calibration-in-the-large and the calibration slope test. MAIN RESULTS AND THE ROLE OF CHANCE: The predictors in the final model were female age, BMI, ethnicity, antral follicle count (AFC), previous live birth, previous miscarriage, cause and duration of infertility. Upon assessing predictive ability, the AUROC curve for the final model and validation cohort was (0.62; 95% confidence interval (CI) 0.61-0.63) and (0.62; 95% CI 0.60-0.64) respectively. Calibration-in-the-large showed a systematic over-estimation of the predicted probability of live birth (Intercept (95% CI) = -0.168 (-0.252 to -0.084), P < 0.001). However, the calibration slope test was not significant (slope (95% CI) = 1.129 (0.893-1.365), P = 0.28). Due to the calibration-in-the-large test being significant we recalibrated the final model. The recalibrated model showed a much-improved calibration. LIMITATIONS, REASONS FOR CAUTION: Our model is unable to account for factors such as smoking and alcohol that can affect IVF/ICSI outcome and is somewhat restricted to representing the ethnic distribution and outcomes for the UK population only. We were unable to account for socioeconomic status and it may be that by having 75% of the population paying privately for their treatment, the results cannot be generalized to people of all socioeconomic backgrounds. In addition, patients and clinicians should understand this model is designed for use before treatment begins and does not include variables that become available (oocyte, embryo and endometrial) as treatment progresses. Finally, this model is also limited to use prior to first cycle only. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study to present a novel, up-to-date model encompassing three readily available prognostic factors; female BMI, ovarian reserve and ethnicity, which have not previously been used in prediction models for IVF outcome. Following geographical validation, the model can be used to build a user-friendly interface to aid decision-making for couples and their clinicians. Thereafter, a feasibility study of its implementation could focus on patient acceptability and quality of decision-making. STUDY FUNDING/COMPETING INTEREST: None.

Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro.

Human oocytes were collected by laparoscopy and fertilized and cultured in vitro. Human chorionic gonadotropin was detected in the medium surrounding two embryos cultured for more than 7 days after fertilization.

The influence of transient hyperprolactinemia on in vitro fertilization in humans.

Hormonal stimulants of ovarian follicular maturation and anesthesia/surgery were examined for their effects on the concentration of plasma and follicular fluid PRL. Forty-seven patients undergoing in vitro fertilization for the treatment of infertility were selected at random for this prospective study. Patients given human menopausal gonadotropin and clomiphene citrate had significantly higher levels of plasma PRL compared to those given clomiphene only. Anesthesia/surgery elevated plasma PRL levels in all patients, by as much as 50-fold and to as high as 7878 mIU/liter. Follicular fluid PRL levels were correlated with preanesthetic plasma PRL concentrations, but the latter were not correlated with plasma 17 beta-estradiol. Elevated plasma or follicular fluid PRL concentrations had no effect on in vitro fertilization of oocytes or embryonic development. Although not significant, the incidence of pregnancy was highest in the group of patients with the lowest preanesthetic plasma PRL levels.

Follicular steroids as a prognosticator of successful fertilization of human oocytes in vitro.

Oocytes were collected by aspiration of preovulatory follicles from 55 women. The preovulatory rise in LH was monitored in urine using the Hi-Gonavis (Mochida Pharmaceuticals) technique. Patients were treated either during the natural cycle or after the induction of ovulation with clomiphene citrate. After collection and culture, the oocytes were inseminated with the spermatozoa of the husband. The levels of progesterone, oestradiol-17 beta and androstenedione in the clear follicular fluid were measured by radioimmunoassay. A multivariate analysis containing these three hormone levels together with two ratios of progesterone with each of the other hormones indicated reasonable discrimination between the oocytes which fertilized and those which remained unfertilized after insemination. The discriminant analysis suggested that the fertilization of the oocytes could have been predicted on the basis of these hormonal profiles with a success rate which exceeded 90%.

Evaluation of the Dinamap continuous blood pressure monitor.

The validity of the Dinamap automated oscillometric blood pressure monitor, an instrument frequently used for continuous monitoring in intensive care settings, was evaluated in comparison to two standard methods of blood pressure measurement. Oscillometric monitoring was compared to random-zero sphygmomanometry in 28 ambulatory subjects, using the instruments in a random order. The oscillometric monitor overestimated random-zero systolic pressure (mean difference = 5.7 mm Hg; P less than 0.001), but did not differ in diastolic pressure (difference = 1.3 mm Hg, P greater than 0.3). Correlation (r) between methods was 0.94 for systolic pressure and 0.83 for diastolic pressure. Repeatability of sequential measures did not differ between methods. To evaluate the oscillometric monitor in the intraoperative setting, it was used simultaneously with a radial artery catheter to measure blood pressure in 14 patients undergoing peripheral vascular or other major surgery. The oscillometric monitor underestimated intra-arterial systolic pressure (mean difference = -9.4 mm Hg; P less than 0.002), but overestimated intraarterial diastolic pressure (difference = 5.7 mm Hg; P less than 0.005). There was no difference in mean arterial pressure (P greater than 0.1). Correlation between methods was 0.88 for systolic, 0.71 for diastolic, and 0.79 for mean arterial pressure. The oscillometric instrument had a sensitivity of 63% and specificity of 97% in detecting intraarterial systolic pressures of 160 mm Hg or greater, and a sensitivity of 89% and specificity of 89% in detecting intraarterial systolic pressures of 100 mm Hg or less. These findings demonstrate differences between the oscillometric monitor and other standard methods of blood pressure measurement.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.

In all, 1210 treatment cycles were divided into three categories for retrospective analysis according to the period of delay between oocyte retrieval (occurring at a fixed time after human chorionic gonadotrophin) and intracytoplasmic sperm injection (ICSI) of <3 h, 3-5 h, >5 h (referred to as 'delayed ICSI'). Three stages from oocyte to the birth of a live baby were identified for statistical analysis, (i) fertilization (2PN zygotes), (ii) cleavage of 2PN zygotes, (iii) transferred embryo to live birth. Stages 1, 2 and 3 were analysed statistically for the three time periods. Chi-square analysis showed no significant effect of delayed ICSI on fertilization (chi(2) = 3.615, P = 0.65), and embryo transfer to birth (chi(2) = 1.840, P = 0.399). The effect on cleavage was significant (chi(2) = 9.625, P = 0.008). However, shorter incubation times produced results which were better than the traditional longer ones. The success rate at the cleavage stage was so high that the marginal advantage had very little effect on the overall process. This study of a substantial patient sample establishes that ICSI on a peri-ovulatory oocyte (<3 h after oocyte retrieval) does not compromise outcome parameters, and that longer periods of incubation (>5 h) do not offer a statistically significant advantage.

Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study.

Twelve patients (17 cycles) who needed >3000 IU of recombinant FSH (rFSH) on previous follicular stimulation attempts for IVF using rFSH (group A) underwent further attempts (a total of 12 cycles) using rFSH supplemented with rLH from day 7 of stimulation (group B). There was no significant difference in the total quantitative administration of rFSH (mean: 4759 versus 4800 IU rFSH), days of stimulation (14.0 versus 12.6), number of M2 oocytes per patient (3.76 versus 4.17) or embryo morphology between group A and group B respectively. However, there was a significant increase in the incidence of fertilization (60.9 versus 86.0%; P = 0.006) and clinical (ongoing) pregnancies (1 versus 6; P = 0.022). The higher incidence of fertilization increased the mean number of embryos transferred per patient, although in this small group the data was not significant (1.75 versus 2.71). This preliminary clinical trial suggests that in poor responders superimposing rLH upon a down-regulated cycle stimulated with rFSH improves relevant outcome data; and this preliminary trial has encouraged the need for a larger and more detailed study.

Endotoxins in culture medium for human in vitro fertilization.

Endotoxins were detected in a few batches of culture medium during the authors' human in vitro fertilization program. Two distinct levels of endotoxins were assayed: greater than 1 ng/ml and less than 1 ng/ml. The source of endotoxin was traced to culture media obtained from a reputable manufacturing company. The incidence of fertilization per patient was not significantly affected by the presence of endotoxins, but fertilization assessed on the overall number of oocytes was significantly reduced (53%) when endotoxin levels were greater than 1 ng/ml compared with an assay negative for endotoxins (66%) (P = 0.047). The percentage of oocytes cleaving after the observation of two pronuclei was not significantly different, but the degree of fragments observed in the conceptus was significantly more severe if the endotoxin level reached 1 ng/ml. In this investigation, the incidence of pregnancy was 8% when the endotoxin level was greater than 1 ng/ml, 30% if less than 1 ng/ml, and 32% if no endotoxins were detected. This study suggests that, although endotoxins may be present in the culture medium at a deleterious level of at least 1 ng/ml, fertilization and cleavage will be obtained, but there will be a significant increase in the incidence of conceptuses with cytoplasmic fragments; this may result in a reduction in the incidence of pregnancy.

Evaluation of high vaginal insemination at oocyte recovery in patients undergoing in vitro fertilization.

The value of high vaginal insemination at the time of oocyte recovery for in vitro fertilization (IVF) has been assessed. A previous study reported a dramatic increase in the incidence of pregnancy in women after high vaginal insemination (53%) compared with those without insemination (23%). Of 306 patients undergoing IVF, 97 (32%) became pregnant and 20 (21%) miscarried. Those patients with tubal damage numbered 187. Sixty (33%) became pregnant and 10 (17%) miscarried. The data were assessed according to the numbers of conceptuses replaced, the age of the patient, and whether a spontaneous luteinzing hormone surge or human chorionic gonadotropin was a trigger for ovulation, each with a significant effect on the outcome of treatment. No significant effect of the use of high vaginal insemination was found.

Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro.

OBJECTIVE: To evaluate subzonal insemination for the procurement of fertilization in infertile males. DESIGN: The spermatozoa of infertile males was used, where possible, for in vitro fertilization (IVF) and subzonal insemination with the same cohort of oocytes obtained from their partner. SETTING: An individual unit in a general hospital, Clinical Nomentana, Rome, Italy. PATIENTS: Males who had repeatedly failed to achieve conception in vitro by IVF or in whom seminal parameters were unacceptable for IVF were treated. INTERVENTIONS: Two hundred twenty-five transvaginal oocyte retrievals were performed. MAIN OUTCOME MEASURES: The incidence of fertilization, cleavage, and pregnancy was evaluated in relation to the numbers of spermatozoa injected and the individual spermatozoa parameters. RESULTS: Fertilization occurred in 39% of patients and 16% of eggs, and 12 clinical pregnancies were established. Fertilization increased with increasing numbers of spermatozoa injected. Fertilization with subzonal insemination was significantly greater (39%) than after in vitro insemination (6%) (P less than 0.0001). CONCLUSIONS: Subzonal insemination improved the incidence of fertilization in this series of patients, and a new classification of spermatozoa based on total motile count aided prognosis for fertilization.

Analysis of 25 infertile patients treated consecutively by in vitro fertilization at Bourn Hall.

Twenty-five infertile women suffering from tubal disorders were treated consecutively by in vitro fertilization over a 14-day period. Follicular growth in 24 of them was stimulated with clomiphene citrate, ovulation being induced by an endogenous surge of luteinizing hormone or an injection of human chorionic gonadotropin. One patient was given tamoxifen and had an endogenous luteinizing hormone surge. One or more oocytes were fertilized, and at least one embryo was replaced in 19 patients. Nine pregnancies were established, and eight infants have been delivered, a pregnancy rate of 36% per laparoscopy and 47% per replacement. A detailed analysis of each patient is presented.

Severe ovarian hyperstimulation syndrome: is it really preventable by prophylactic intravenous albumin?

OBJECTIVE: To evaluate the effectiveness of i.v. albumin in preventing severe ovarian hyperstimulation syndrome (OHSS) in patients at risk. DESIGN: Retrospective review and data analysis. SETTING: University-based tertiary referral center for assisted reproductive technologies (ART). PATIENT(S): Sixty women at high risk of developing severe OHSS after superovulation for ART. INTERVENTION(S): One liter of albumin (4.5%) administered i.v. during oocyte retrieval and immediately afterward. RESULT(S): Of the 60 women who had prophylactic i.v. albumin, 5 (8%) developed severe OHSS, which led to hospitalization. Eight (13%) developed moderate OHSS. Forty-seven (78%) did not develop any symptoms. Four of the 5 women who developed severe OHSS had ET and 3 of them (75%) were pregnant (1 twin and 2 singletons). CONCLUSION(S): Intravenous albumin administered at oocyte retrieval does not prevent the occurrence of severe OHSS, especially in cases associated with pregnancy. It is important that clinicians are not lured into a false sense of security by the early report, full of promise, on the use of i.v. albumin to prevent severe OHSS.

Gamma-aminobutyric acid (GABA) A and B receptors mediate the stimulatory effects of GABA on the human sperm acrosome reaction: interaction with progesterone.

OBJECTIVE: To evaluate which gamma-aminobutyric acid (GABA) receptor mediates the stimulatory effects of this neurotransmitter on the human sperm acrosome reaction, and to examine the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA(A) receptor. DESIGN: Prospective study. SETTING: A university clinic of andrology. PATIENT(S): Men with normal sperm analysis parameters. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The acrosome reaction of motile spermatozoa. RESULT(S): The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA(A) receptor antagonist, and only partially by saclofen, a GABA(B) receptor antagonist. Accordingly, muscimol, a GABA(A) receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen, a GABA(B) receptor agonist, was less effective. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome-reacted spermatozoa compared with progesterone alone. However, this increase was less than a simple addition of effects, suggesting that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the ability of progesterone to induce acrosome reaction was tested in the presence of bicuculline, which suppressed the stimulatory effects of progesterone. Given that the GABA(A) receptor is linked to the chloride channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Picrotoxin completely suppressed the acrosome reaction induced by progesterone and only partially suppressed that caused by GABA. CONCLUSION(S): gamma-Aminobutyric acid stimulated the acrosome reaction in human spermatozoa, acting mainly through the GABA(A) receptor and to a lesser extent through the GABA(B) receptor. Progesterone interacted with the GABA(A) receptor to induce the acrosome reaction, and the functional integrity of the chloride channel was vital for this effect.

Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study.

The survival and implantation capacity of cryopreserved cleaving (5-cell to 10-cell) human embryos and expanded blastocysts was compared. Twice as many cleaving embryos were frozen as were expanding blastocysts because of the low developmental potential of human embryos in vitro. However, significantly more expanded blastocysts survived cryopreservation than cleaving embryos, and relatively more pregnancies were established by the replacement of thawed blastocysts than by the replacement of thawed cleaving embryos. Cleaving embryos from 26 women were thawed; 17 had thawed embryos replaced, and 4 subsequently became pregnant. Expanded blastocysts were thawed from 23 other women; 15 had thawed blastocysts replaced, and 8 subsequently became pregnant. The pregnancy of one patient in each group aborted; both patients were over 40 years of age. It is estimated that by maintaining the current policy of replacing three fresh embryos and freezing any remaining embryos when they reach blastocyst stage, the total incidence of pregnancy would increase by 3%.

Subzonal insemination for the alleviation of infertility.

Methods were developed to facilitate the use of subzonal insemination to achieve fertilization in vitro. A clinical trial was undertaken in those patients having previously failed to achieve fertilization by in vitro fertilization, and in those presenting with severe oligospermia/oligoasthenospermia. From 85 patients, 585 oocytes were obtained. Of these, 0.3% had been "activated" parthenogenetically in vivo, 369 (72%) at metaphase II underwent subzonal insemination, 15% were fertilized, and 0.5% had three pronuclei. Thirty-eight percent of the patients had fertilization with 36% having a replacement. One conceptus was replaced in 19 patients, two conceptus in 8 patients, and three in 4 patients. A twin and two singleton pregnancies were established.

Predicting empty follicle syndrome.

OBJECTIVE: To determine a simple predictive test for empty follicle syndrome before oocyte retrieval. DESIGN: Retrospective correlation analysis. SETTING: Patients attending Nottingham University Research and Treatment Unit, a tertiary, university-based assisted reproductive technologies (ART) program between April 1, 1994 and March 31, 1995. PATIENT(S): Six women in whom no oocytes were retrieved after superovulation for ART (empty follicle syndrome) were compared with 11 women with successful oocyte retrieval. INTERVENTION(S): Subcutaneous buserelin acetate plus IM hMG and hCG were used for superovulation. Oocyte retrieval was transvaginal and ultrasound guided. MAIN OUTCOME MEASURE(S): Ultrasound measurement of follicular growth and serum E2 levels during superovulation. Serum beta-hCG levels before and 36 hours after hCG administration IM. Number of oocytes retrieved. RESULT(S): Before hCG administration, beta-hCG was not detectable in the serum. The serum beta-hCG 36 hours after hCG was 209 +/- 16.7 mIU/mL (conversion factor to SI units, 1.0; mean +/- SEM, range 106 to 290 mIU/mL) in women with successful oocyte retrieval and 4 +/- 1.8 mIU/mL (range 0 to 9 mIU/mL) in empty follicle syndrome. This difference was significant. CONCLUSION(S): Empty follicle syndrome is associated with very low bioavailability of beta-hCG and can be predicted by measuring serum beta-hCG level 36 hours after IM hCG administration.

In vitro fertilization: a treatment for male infertility.

Three-quarters of 41 women whose husbands were the cause of infertility in the couple had one or more embryos replaced after in vitro fertilization. One-third became pregnant despite their long history of infertility; the incidence of pregnancy was 45% after one or more replacements of embryos. In a group of 61 couples where both partners were infertile, 26% became pregnant. These data indicate that the lower chance of fertilization with poor semen is balanced by a higher chance of pregnancy in women with a normally functioning reproductive system. Oligospermia, asthenospermia, teratospermia, and autoimmunity are among the many forms of male infertility which can now be successfully treated by in vitro fertilization. Overall, 57% of the oocytes were fertilized, and almost 50% of men with very low numbers of active spermatozoa (less than or equal to 0.5 X 10(6)/ml motile spermatozoa) were successful in establishing pregnancy. Two semen parameters impaired fertilization most: seminal inflammatory cells and low progressive activity. The collection of split ejaculates and the careful preparation of spermatozoa, by sedimentation and layering methods, proved to be beneficial, improving sperm motility and raising the chance of fertilization.

In vitro fertilization using cryopreserved donor semen in cases where both partners are infertile.

The incidence of pregnancy after in vitro fertilization (IVF) was studied in a group of 38 couples (55 cycles) where both partners were infertile. Cryopreserved donor semen (IVF-D) was used in all cycles. Results were compared with those in a control group of couples where the husband's semen was considered normal and only the wife was infertile. No significant differences were found between the IVF-D and control groups in the incidence of fertilization (80% versus 72%), pregnancy per cycle (33% versus 29%), and abortion (18% versus 20%), despite the considerably lower percentage of motile spermatozoa in the IVF-D group. Forty percent of patients, each treated unsuccessfully with at least 12 artificial inseminations with donor semen, became pregnant after one or two IVF-D cycles. It is concluded that IVF with frozen donor semen is a beneficial treatment for couples where both partners are infertile.