Detection of virulence associated genes in Streptococcus agalactiae isolated from bovine mastitis.

Background: Mastitis is one of the most expensive diseases in the dairy industry. It causes heavy monetary losses by decreasing milk production and treatment cost. Streptococcus agalactiae, the cause of contagious bovine mastitis, possesses various virulence factors that contribute to pathogenicity. Aims: The main aim of the study was to evaluate the distribution of virulence genes of S. agalactiae. Methods: In the current work, 98 Streptococcus species were isolated from 320 milk samples, collected from Veterinary Clinical Complex, Junagadh. Out of the isolates, 42 S. agalactiae isolates were used for virulence genes detection. Results: All Streptococcus spp. were confirmed at genus level by targeting tuf gene, and S. agalactiae was identified at species level by targeting 16S rRNA gene. For virulence gene detection, scpB, cfb, and cylE genes were targeted. Of 42 S. agalactiae isolates, 15, 16, and 10 isolates possessed scpB, cfb, and cylE genes, respectively. Conclusion: This study aids us to know virulence characteristics and mechanisms responsible for the development of new strains in mastitis epidemiology in response to prevention and control strategies.

Preliminary report of intraovarian injections of autologous platelet-rich plasma (PRP) in extremely poor prognosis patients with only oocyte donation as alternative: a prospective cohort study.

STUDY QUESTION: Does intraovarian injection of platelet-rich plasma (PRP) change ovarian function in patients with extremely low functional ovarian reserve (LFOR) who, otherwise, would likely only have a chance of pregnancy through third-party oocyte donation? SUMMARY ANSWER: No clinically significant effects of PRP treatment on ovarian function were observed over 1 year of follow-up. WHAT IS KNOWN ALREADY: Several investigators have reported improved responses to ovulation induction after treatment with PRP. However, previous published reports have involved, at most, only small case series. Whether PRP actually improves ovarian performance is, therefore, still unknown. PRP is nevertheless widely offered as an 'established' fertility treatment, often under the term 'ovarian rejuvenation'. STUDY DESIGN SIZE DURATION: We are reporting a prospective cohort study of 80 consecutive patients at ages 28-54 with LFOR, defined by anti-Müllerian hormone <1.1 ng/ml, FSH >12 mIU/ml or at least one prior IVF cycle with ≤3 oocytes within 1 year. The women were followed for 1 year after an intraovarian PRP procedure. PARTICIPANTS/MATERIALS SETTING METHODS: PRP (1.5 ml) was injected into the cortex of ovaries with an average of 12 injections per ovary. Study participants were followed every 3 days for 2 weeks after PRP treatment with estradiol and FSH measurements and vaginal ultrasound to observe follicle growth and thereafter followed weekly. Beginning 1 month after their PRP treatment, participants underwent one or more cycles of ovarian stimulation for IVF. Outcome measures were endocrine response, and numbers of oocytes and embryos produced in response to a maximal gonadotropin stimulation before and after PRP treatment. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, women failed to demonstrate statistically significant outcome benefits from intraovarian PRP. However, two 40-year-old very poor-prognosis patients, with prior failed IVF cycles that never reached embryo transfer at other centers, achieved pregnancy, resulting in an ongoing pregnancy rate of 4.7% among patients who, following PRP, produced at least one oocyte (n = 42). LIMITATIONS REASONS FOR CAUTION: As an observational study of patients who performed poorly in past ovarian stimulation cycles, the improvement may be accounted for by regression to the mean. Similar considerations may also explain the occurrence of the two pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates that, even in extremely poor prognosis patients due to LFOR, sporadic pregnancies are possible. The study, however, does not allow for the conclusion that those pregnancies were the consequence of PRP treatments. A case series, indeed, does not allow for such conclusions, even if results are more suggestive than here. This registered study, therefore, must be viewed as a preliminary report, with further data expected from this study but also from two other prospectively randomized ongoing registered studies with more controlled patient selection. STUDY FUNDING/COMPETING INTERESTS: This work was supported by intramural funds from The Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several US patents. Some of these patents relate to pre-supplementation of hypo-androgenic infertile women with androgens, such as dehydroepiandrosterone and testosterone and, therefore, at least peripherally relate to the subject of this manuscript. They, as well as D.F.A., have also received research support, travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC. E.M. has no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NCT04275700.

IVF outcomes of embryos with abnormal PGT-A biopsy previously refused transfer: a prospective cohort study.

STUDY QUESTION: What are the outcomes for patients who choose to move embryos diagnosed as abnormal by preimplantation genetic testing for aneuploidy (PGT-A) to a new institution for transfer after the diagnosing institution refused to transfer them? SUMMARY ANSWER: Many patients seek to have selected embryos with PGT-A abnormal trophectoderm biopsies transferred recognizing that these embryos can still offer a chance of pregnancy and live birth. WHAT IS KNOWN ALREADY: : PGT-A is a widely practiced method of selecting embryos for transfer based on biopsy of a few cells. Many clinical practices refuse to transfer PGT-A abnormal embryos even when there are no other 'normal' embryos available. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort of 69 couples who, since 2014, moved a total of 444 PGT-A abnormal embryos previously refused transfer at their parent institutions to our practice. Among these, 50 patients have, thus far, undergone 57 transfer cycles of 141 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos diagnosed at other institutions by PGT-A as abnormal (mostly using next generation sequencing) were moved to our academically affiliated private fertility and research center in New York City. Female age at retrieval was 41.35 ± 3.98 years, 74% were Caucasian, 12% Asian and 10% were of African descent. All embryos identified as PGT-A abnormal among prospectively identified couples were recorded in our center's registry. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 144 embryos transferred 102 (72.3%) had only 1 or 2 chromosomal abnormalities, 30 (21.3%) had 3 or more and 9 (6.4%) were 'undiagnosed' because of degraded DNA, yet still had been refused transfer. Transfer of PGT-A abnormal embryos resulted in 8 live births, 11 miscarriages and no voluntary terminations. One child was born with a segmental duplication and required repair of coarctation of the aorta as a newborn. Many couples with only PGT-A abnormal embryos are willing to have their PGT-A abnormal embryos transferred and such transfers can result in the establishment of ongoing euploid pregnancies and live births. LIMITATIONS, REASONS FOR CAUTION: Findings in this case series represent couples who chose to have their embryos transferred after having been refused transfer elsewhere and may not be representative of the wider population of couples undergoing IVF with PGT-A in general. Not all abnormal phenotypes present in the immediate postnatal period so it will be important to continue to follow the development of these children. WIDER IMPLICATIONS OF THE FINDINGS: PGT-A can result in a clinics refusal to transfer embryos with abnormal PGT-A biopsies, even those with mosaic findings, consequently large numbers of infertile women are prematurely advised that their only chance of motherhood is through third-party egg-donation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funds from the Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several U.S. patents. One of these patents (US Patent# 7,615,544) relates to pre-supplementation of hypo-androgenic infertile women with androgens, such as DHEA and testosterone and, therefore, at least peripherally related to the subject of this manuscript. N.G. and D.F.A. also received travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC. TRIAL REGISTRATION NUMBER: N/A.

The ovarian sensitivity index is predictive of live birth chances after IVF in infertile patients.

STUDY QUESTION: Does the ovarian sensitivity index (OSI) predict embryo quality, pregnancy and live birth in patients undergoing FSH/hMG stimulation for IVF? SUMMARY ANSWER: The OSI is predictive of pregnancy and live birth in older women with a more unfavorable prognosis undergoing FSH/hMG stimulation for IVF. WHAT IS KNOWN ALREADY: The OSI was previously reported to reflect gonadotrophin requirements among high, normal and poor responders and to predict pregnancy potential in younger patients undergoing ovarian stimulation with FSH. STUDY DESIGN SIZE DURATION: A retrospective cohort study that included 1282 women undergoing IVF with FSH/hMG stimulation was carried out between January 2010 and December 2016. PARTICIPANTS/MATERIALS SETTING METHODS: We evaluated 1282 women who underwent fertility treatment with FSH/hMG stimulation and oocyte retrieval at an academically affiliated private fertility center. OSI was calculated as (oocytes ×1000)/total gonadotrophin dose and grouped into two classes based on a receiver operating characteristic (ROC) curve analysis of a randomly selected development sample comprising one-third of the cycles. The remaining cycles comprised the validation group. ROC curves were also used to compare the predictive value of OSI to that of baseline FSH and anti-Müllerian hormone (AMH). Logistic regression models evaluated the effect of high (OSI >0.83) and low (OSI ≤0.83) on clinical pregnancy and live birth in the validation group. Models were adjusted for female age, baseline FSH, AMH and oocyte yield and gonadotrophin dose. MAIN RESULTS AND THE ROLE OF CHANCE: Women presented with a mean ±SD age of 38.6 ± 5.4 years and showed median AMH levels of 0.65 (95% CI 0.61-0.74) ng/ml. They received 5145 ± 2477 IU of gonadotrophins and produced a median 5.2 (95% CI 5.0-5.5) oocytes. Pregnancy and live birth rates per oocyte retrieval for all women were 20.6% and 15.8%, respectively. Patients with higher OSI (less gonadotrophin required per oocyte retrieved) produced significantly more high-quality embryos than patients with low OSI (3.5 (95% CI 3.2-3.8) versus 0.6 (95% CI 0.5-0.7) (P = 0.0001)) and demonstrated higher pregnancy (23.2% versus 9.7%) and live birth rates (8.8% versus 5.3%) than their counterparts (P = 0.0001 and P = 0.0001, respectively). After adjustments for age, baseline AMH and FSH, total gonadotrophin dosage and oocyte yield, an OSI >0.83 was associated with greater odds of pregnancy (odds ratio 2.12, 95% CI 1.30-3.45, P < 0.003) and live birth (odds ratio 1.91, 95% CI 1.07-3.41, P < 0.028). LIMITATIONS REASONS FOR CAUTION: The results may not be applicable to women with excellent pregnancy potential or FSH-only stimulation. WIDER IMPLICATIONS OF THE FINDINGS: The predictive capacity of OSI for embryo quality, pregnancy and live birth, which is independent of AMH or FSH, may help in counseling patients about their pregnancy potential and live birth chances. STUDY FUNDING/COMPETING INTERESTS: Intramural funding from the Center for Human Reproduction and the Foundation for Reproductive Medicine. A.W., V.A.K., D.F.A., D.H.B. and N.G. have received research grant support, travel funds and speaker honoraria from various pharmaceutical and medical device companies: none, however, related to the topic presented here. D.H.B. and N.G. are listed as inventors on already awarded and still pending US patents, claiming beneficial effects on diminished ovarian reserve and embryo ploidy from dehydroepiandrosterone supplementation. TRIAL REGISTRATION NUMBER: N/A.

The 2019 PGDIS position statement on transfer of mosaic embryos within a context of new information on PGT-A.

BACKGROUND: A recently published Position Statement (PS) by the Preimplantation Genetics Diagnosis International Society (PGDIS) regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF) contained inaccuracies and misrepresentations. Because opinions issued by the PGDIS have since 2016 determined worldwide IVF practice, corrections appear of importance. METHODS: The International Do No Harm Group in IVF (IDNHG-IVF) is a spontaneously coalesced body of international investigators, concerned with increasing utilization of add-ons to IVF. It is responsible for the presented consensus statement, which as a final document was reached after review of the pertinent literature and again revised after the recent publication of the STAR trial and related commentaries. RESULTS: In contrast to the PGDIA-PS, we recommend restrictions to the increasing, and by IVF centers now often even mandated, utilization of PGT-A in IVF cycles. While PGT-A has been proposed as a tool for achieving enhanced singleton livebirth outcomes through embryo selection, continued false-positive rates and increasing evidence for embryonic self-correction downstream from the testing stage, has led IDNHG-IVF to conclude that currently available data are insufficient to impose overreaching recommendations for PGT-A utilization. DISCUSSION: Here presented consensus offers an alternative to the 2019 PGDIS position statement regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF). Mindful of what appears to offer best outcomes for patients, and in full consideration of patient autonomy, here presented opinion is based on best available evidence, with the goal of improving safety and efficacy of IVF and minimizing wastage of embryos with potential for healthy births. CONCLUSIONS: As the PGDIS never suggested restrictions on clinical utilization of PGT-A in IVF, here presented rebuttal represents an act of self-regulation by parts of the IVF community in attempts to control increasing utilization of different unproven recent add-ons to IVF.

Age, body weight and ovarian function affect oocyte size and morphology in non-PCOS patients undergoing intracytoplasmic sperm injection (ICSI).

The size of oocytes was previously reported to be smaller in obese women with polycystic ovary syndrome (PCOS). In the present prospective cohort study, we sought to determine whether oocyte size and morphology are associated with patient characteristics in non-PCOS women. Oocyte and oolemmal diameter were measured, enlarged perivitelline space (PVS) and ooplasmic granulation were assessed in 308 MII oocytes from 77 IVF/ICSI couples. Statistical analysis was undertaken using SAS version 9.4 (SAS institute Inc., USA). Continuous values are presented as mean ± SD and compared using a two-sample t-test or Mann-Whitney U test as appropriate. Categorical parameters are presented as proportions and compared using a Fisher exact test. Logistic and linear regression models were used to control for the effect of age for categorical and continuous variables respectively. P-value < 0.05 was considered statistically significant. Patients presented with a mean age of 40.3±5.0 years, had a BMI of 25.1±6.1 kg/m2, median AMH levels of 0.6 ng/ml and produced a median of 4 oocytes. Mean total oocyte diameter was 163.2±7.4 µm (range 145.8-182.1 µm), while oolemmal diameter was 109.4±4.1 µm (range 98.5-122.3 µm). After adjusting for age and ovarian reserve increasing BMI was associated with decreased total oocyte diameter (p<0.05). Total oocyte diameter was also inversely associated with AMH levels (p = 0.03) and oocyte yield (p = 0.04). In contrast to total oocyte diameter, oolemmal diameter was not related to patient characteristics. Younger women and those with large oocyte yields demonstrated fewer oocytes with ooplasmic granulation (p<0.05 and p = 0.01). After adjustments for age, ooplasmic granulation was also less frequently observed in oocytes from women with higher AMH (p = 0.03) and increasing BMI (p<0.01). Fertilization was more likely in oocytes with larger oolemmal diameter (p = 0.008). Embryos from oocytes with larger total and ooplasmic diameters were more likely to be transferred or frozen (p = 0.004 and p = 0.01). In non-PCOS infertile women, BMI and ovarian function relate to total oocyte diameter. These results expand on previously observed associations between oocyte size and BMI in women with PCOS. They indicate the importance of detailed oocyte assessments, which may aid the currently used criteria for embryo selection and help to better understand how oocyte status is associated with later embryo development.

Worldwide decline of IVF birth rates and its probable causes.

With steadily improving pregnancy and live birth rates, IVF over approximately the first two and a half decades evolved into a highly successful treatment for female and male infertility, reaching peak live birth rates by 2001-2002. Plateauing rates, thereafter, actually started declining in most regions of the world. We here report worldwide IVF live birth rates between 2004 and 2016, defined as live births per fresh IVF/ICSI cycle started, and how the introduction of certain practice add-ons in timing was associated with changes in these live birth rates. We also attempted to define how rapid worldwide 'industrialization' (transition from a private practice model to an investor-driven industry) and 'commoditization' in IVF practice (primary competitive emphasis on revenue rather than IVF outcomes) affected IVF outcomes. The data presented here are based on published regional registry data from governments and/or specialty societies, covering the USA, Canada, the UK, Australia/New Zealand (combined), Latin America (as a block) and Japan. Changes in live birth rates were associated with introduction of new IVF practices, including mild stimulation, elective single embryo transfer (eSET), PGS (now renamed preimplantation genetic testing for aneuploidy), all-freeze cycles and embryo banking. Profound negative associations were observed with mild stimulation, extended embryo culture to blastocyst and eSET in Japan, Australia/New Zealand and Canada but to milder degrees also elsewhere. Effects of 'industrialization' suggested rising utilization of add-ons ('commoditization'), increased IVF costs, reduced live birth rates and poorer patient satisfaction. Over the past decade and a half, IVF, therefore, has increasingly disappointed outcome expectations. Remarkably, neither the profession nor the public have paid attention to this development which, therefore, also has gone unexplained. It now urgently calls for evidence-based explanations.

A pilot cohort study of granulocyte colony-stimulating factor in the treatment of unresponsive thin endometrium resistant to standard therapies.

STUDY QUESTION: Is thin endometrium unresponsive to standard treatments expandable by intrauterine perfusion with granulocyte colony-stimulating factor (G-CSF)? SUMMARY ANSWER: This cohort study is supportive of the effectiveness of G-CSF in expanding chronically unresponsive endometria. WHAT IS KNOWN ALREADY: In a previous small case series, we reported the successful off-label use of G-CSF in four consecutive patients, who had previously failed to expand their endometria beyond 6.9 mm with the use of standard treatments. STUDY DESIGN, SIZE AND DURATION: In a prospective observational cohort pilot study over 18 months, we described 21 consecutive infertile women with endometria <7 mm on the day of hCG administration in their first IVF cycles at our center. All previous cycles using traditional treatments with estradiol, sildenafil citrate (Viagra™) and/or beta-blockers had been unsuccessful. G-CSF (Nupogen™) was administered per intrauterine catheter by slow infusion before noon on the day of hCG administration. If the endometrium had not reached at least a 7-mm within 48h, a second infusion was given following oocyte retrieval. Primary and secondary main outcomes were an increase in endometrial thickness and clinical pregnancy, respectively. Endometrial thickness was assessed by vaginal ultrasound at the most expanded area of the endometrial stripe. PARTICIPANTS/MATERIALS, SETTINGS AND METHOD: This study was uncontrolled, each patient serving as her own control in a prospective evaluation of endometrial thickness. The mean ± SD age of the cohort was 40.5 ± 6.6 years, gravidity was 1.8 ± 2.1 (range 0-7) and parity was 0.4 ± 1.1 (range 0-4); 76.2% of women had, based on age-specific FSH and anti-Müllerian hormone, an objective diagnosis of diminished ovarian reserve and had failed 2.0 ± 2.1 prior IVF cycles elsewhere. MAIN RESULTS AND THE ROLE OF CHANCE: With 5.2 ± 1.9 days between G-CSF perfusions and embryo transfers, endometrial thickness increased from 6.4 ± 1.4 to 9.3 ± 2.1 mm (P < 0.001). The Δ in change was 2.9 ± 2.0 mm, and did not vary between conception and non-conception cycles. A 19.1% ongoing clinical pregnancy rate was observed, excluding one ectopic pregnancy. LIMITATIONS AND REASONS FOR CAUTION: Small sample size (but a highly selected patient population) in an uncontrolled cohort study and in unselected first IVF cycles at our center. WIDER IMPLICATIONS OF THE FINDINGS: This pilot study supports the utility of G-CSF in the treatment of chronically thin endometrium and suggests that such treatment will, in very adversely affected patients, result in low but very reasonable clinical pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Foundation for Reproductive Medicine, New York, New York, USA, a not-for-profit research foundation and intramural grants from the Center for Human Reproduction (CHR)-New York. N.G. and D.H.B. are members of the board of the Foundation for Reproductive Medicine. N.G. is owner of CHR-New York, where the study was conducted. N.G. and D.H.B. have been recipients of research awards, travel grants and speaker honoraria from various pharmaceutical and medical device companies. None of these companies was, however, in any way associated with the materials and the manuscript presented here. N.G. and D.H.B. are listed as co-inventors on a number of awarded and still pending U.S. patents, none related to the materials presented here. N.G. is on the board of a medically related company, not in any way associated with the data presented here.

Unexplained infertility: does it really exist?

Recent medical literature has quite extensively addressed the use of various terminologies within the field of reproductive medicine. This discussion has, however, so far overlooked the fact that one of the most frequently made diagnosis, so-called unexplained infertility (UI), not only didactically but, even more importantly, clinically, appears unsustainable as an independent diagnosis. The arguments in support of such a contention are manifold. The diagnosis of UI is highly subjective. It is dependent on which diagnostic tests have been performed (or have been omitted) and at what level of quality. Paradoxically, a diagnosis of UI will, therefore, be more often reached if the diagnostic workup is incomplete or of poor quality. Supported by evidence from the literature, the argument is made that the conditions, most frequently misdiagnosed as UI, are endometriosis, tubal infertility (especially distal and peritubal disease), premature ovarian ageing and immunological infertility. Because of the obvious unreliability of a diagnosis of UI and the widely reported unevenness in diagnostic criteria, we recommend the abandonment of UI as a formal infertility diagnosis. Better efforts to reach infertility diagnoses more accurately should improve the diagnostic accuracy of hitherto frequently missed diagnoses, which often falsely have led to a diagnosis of UI.

Habitual tea consumption and risk of osteoporosis: a prospective study in the women's health initiative observational cohort.

The purpose of this study was to prospectively investigate associations of habitual drinking of regular tea with bone mineral density and fracture risk. Study participants were a multiethnic postmenopausal cohort (n = 91,465) from the nationwide Women's Health Initiative Observational Study. These women were recruited in the United States and aged 50-79 years at the time of enrollment (1994-1998). The average follow-up time was 4.1 years. Habitual consumption of regular tea was assessed with a structured questionnaire at baseline. Clinical fractures during the follow-up were reported in questionnaires, and hip fractures were further confirmed by reviewing medical records. Bone mineral density measurements were conducted among a subgroup of women (n = 4,979) at three Women's Health Initiative bone mineral density centers using dual-energy x-ray absorptiometry. Multivariate analyses suggested a positive trend of increased total body bone mineral density with tea drinking (p < 0.05). However, results from the Cox proportional hazard models did not show any significant association between tea drinking and the risk of fractures at the hip and forearm/wrist. In conclusion, the results from this study indicate that the effect of habitual tea drinking on bone density is small and does not significantly alter the risk of fractures among the US postmenopausal population.

Modified open laparoscopy through placement of an optical surgical obturator.

OBJECTIVE: To describe a new technique for open laparoscopy. DESIGN: Prospective case series. SETTING: Ambulatory surgical unit. PATIENT(S): Fifty-six women undergoing laparoscopic sterilization. INTERVENTION(S): Intra-abdominal placement of an optical surgical obturator. MAIN OUTCOME MEASURE(S): Evaluation of surgical technique. RESULT(S): The technique was completed successfully in all cases. CONCLUSION(S): This new surgical approach may assist surgeons in avoiding inadvertent injuries.