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Research Question: Do platelet-rich plasma (PRP) products, specifically human platelet lysate (hPL) and umbilical cord plasma, enhance vascularization and follicular survival in human ovarian tissue transplanted to immunodeficient mice?, Design: Human ovarian tissue was transplanted to subcutaneous pockets in nude mice, followed by daily injections for 6 days of PRP or saline at the transplantation sites. After a grafting period of 3 and 6 days, vascularization was assessed using CD-31 quantification, and gene expression of angiogenic markers (VEGF/Vegf) together with apoptosis-related genes (BAX/BCL-2), oxidative stress markers (HMOX-1/Hmox-1) and pro-inflammatory markers (Il-1β/Il-6/Tnf-α) was quantitively analysed. Follicle density was analysed in the grafts after 4 weeks. Additionally, a pilot study was conducted exploring the suitability of ultrasound scanning for assessing survival and vascularization in ovarian tissue xenografted to mice., Results: Although there was a significant increase in the CD-31 area from day 3 to day 6 post-grafting, there were no significant differences between the hPL and control groups. Gene expression analysis revealed significant down-regulation of VEGF from day 3 to day 6 for both the hPL and control groups, and significant up-regulation of BAX/BCL-2 in the hPL group compared with the controls. The follicle density showed no significant differences in the hPL group and UCP groups compared with the controls. Furthermore, ultrasound biomicroscopy provided valuable insights into graft morphology, necrotic areas and blood flow, suggesting its potential as a monitoring tool., Conclusions: Despite the angiogenic properties of PRP, this study was unable to demonstrate a significant impact of hPL on vascularization or of hPL and UCP on follicular survival in xenotransplanted human ovarian tissue., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Research Question: Does splitting the human chorionic gonadotrophin (HCG) support in IVF cycles triggered by a gonadotrophin-releasing hormone agonist result in a better progesterone profile?, Design: Randomized controlled three-arm study, performed at the Fertility Clinic, Odense University Hospital, Denmark. Patients with 12-25 follicles ≥12 mm were randomized into three groups: Group 1 - ovulation triggered with 6500 IU HCG; Group 2 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1500 IU HCG on the day of oocyte retrieval (OCR); and Group 3 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1000 IU HCG on the day of OCR and 500 IU HCG on OCR + 5. All groups received 180 mg vaginal progesterone. Progesterone concentrations were analysed in eight blood samples from each patient., Results: Sixty-nine patients completed the study. Baseline and laboratory data were comparable. Progesterone concentration peaked on OCR + 4 in Groups 1 and 2, and peaked on OCR + 6 in Group 3. On OCR + 6, the progesterone concentration in Group 2 was significantly lower compared with Groups 1 and 3 (P = 0.003 and P < 0.001, respectively). On OCR + 8, the progesterone concentration in Group 3 was significantly higher compared with the other groups (both P<0.001). Progesterone concentrations were significantly higher in Group 3 from OCR + 6 until OCR + 14 compared with the other groups (all P ≤ 0.003). Four patients developed ovarian hyperstimulation syndrome in Group 3., Conclusion: Sequential HCG support after a GnRH agonist trigger provides a better progesterone concentration in the luteal phase., (Copyright © 2023. Published by Elsevier Ltd.)

Research Question: How do platelet-rich plasma products like human platelet lysate (HPL) and umbilical cord plasma (UCP) affect the growth and survival of isolated human pre-antral follicles in vitro?, Design: Human pre-antral follicles (n = 724; mean diameter: 75 µm; range: 46-237 µm) were isolated from ovarian medulla donated by 14 patients undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with 5% fetal bovine serum (FBS) (n = 171), 2.5% human serum albumin (HSA) (n = 159), 5% HPL (n = 223) or 5% UCP (n = 171)., Results: The survival probability was significantly higher in the group supplemented with HPL (80%) compared with the other three groups: FBS (54%, P < 0.001); HSA (63%, P = 0.004) and UCP (29%, P < 0.001). Surviving follicles in the UCP group had less defined follicular membranes and decompacted granulosa cell layers. The median growth of surviving follicles was significantly (P < 0.001) larger in the HPL group (73 µm) compared with any of the other three groups: HSA (43 μm); FBS (40 μm) UCP (54 μm). A descriptive analysis of follicular secretion of anti-Müllerian hormone and oestradiol did not reveal any difference between the groups. The detectability of follicular genes was high for AR (100%), AMHR2 (100%) and FSHR (76%), whereas few follicles expressed LHR (20%)., Conclusion: Human platelet lysate significantly improved survival and growth of cultured human pre-antral follicles compared with FBS, HSA and UCP. The use of HPL is a valuable improvement to culture human pre-antral follicles but further studies will have to prove whether the superiority of HPL translates into better quality oocytes., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Research Question: Is there an association between the ovulation trigger dose of human chorionic gonadotrophin (HCG) and endogenous progesterone production during the luteal phase?, Design: This randomized controlled four-arm study, at the Fertility Clinic, Odense University Hospital, Denmark, included women undergoing gonadotrophin-releasing hormone (GnRH) antagonist IVF treatment with ≤11 follicles ≥12 mm. Group 1-3 were triggered with 5000 IU, 6500 IU or 10,000 IU HCG, respectively, receiving 17α-hydroxyprogesterone caproate intramuscularly for luteal-phase support (LPS) to measure endogenous progesterone production. Group 4 received 6500 IU HCG trigger and vaginal progesterone. During the study, the 5000 IU and 10,000 IU HCG groups were switched from urinary to recombinant HCG, as urinary HCG was removed from market. Eight blood samples were drawn during the luteal phase., Results: Ninety-four participants completed the study. There was a significant positive association between the HCG trigger dose and the progesterone at 8 days (P < 0.001), 10 days (P < 0.001) and 14 days (P < 0.001) post-oocyte retrieval. Comparing the groups individually revealed a significant difference in progesterone concentration between low and high trigger doses at 4 days (P = 0.037) and 8 days (P = 0.007) post-oocyte retrieval and between all intervention groups at oocyte retrieval + 6 days: group 1 and 2 (P = 0.011), group 2 and 3 (P = 0.042) and group 1 and 3 (P < 0.001). Higher HCG trigger dose increased the progesterone from the individual follicle., Conclusions: Increasing HCG trigger doses significantly increased endogenous progesterone concentration during the mid-late luteal phase., (Copyright © 2022. Published by Elsevier Ltd.)

Research Question: What is the impact of systemic FSH concentrations during ovarian stimulation for IVF/intracytoplasmic sperm injection on systemic progesterone concentrations in the late follicular phase?, Design: Post-hoc analysis of a previously performed randomized controlled trial (RCT) performed between November 2017 and February 2020 in a tertiary IVF centre. The RCT included patients with infertility undergoing ovarian stimulation in a gonadotrophin-releasing hormone (GnRH) antagonist protocol. The GnRH antagonist was administered at 08:00 h and recombinant FSH at 20:00 h. Ultrasound and blood tests were performed 3-5 h after the GnRH antagonist., Results: The subgroup analysis comprised 105 patients. Systemic FSH concentrations increased from Day 2/3 until initiation of GnRH antagonist and remained constant until the day of trigger (DoT). The total group was split according to the median FSH DoT concentration (12.95 IU/l; Group A <12.95 IU/l; Group B ≥12.95 IU/l). Significant differences, with the higher concentrations in Group B, were found for: systemic FSH concentration on Day 2/3 (P = 0.04), total gonadotrophin dosage (P = 0.03), progesterone on DoT (P = 0.001) and progesterone per follicle (P = 0.004). In the total group, systemic DoT FSH concentration was statistically significantly positively correlated with the DoT progesterone concentration and the ratio of progesterone per follicle (ρ = 0.37 and 0.38, respectively, both P < 0.001). No significant correlations were seen between the systemic DoT FSH concentration and the number of retrieved oocytes., Conclusion: While ovarian response seems to be independent from the systemic FSH concentrations on the DoT, high concentrations of circulatory FSH augment the production of progesterone., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Research Question: What is the frequency of morphological dysmorphisms in immature human oocytes collected ex vivo from small antral follicles and matured in vitro?, Design: Human ovaries (n = 56) were excised for ovarian tissue cryopreservation (OTC). None of the patients had received exogenous gonadotrophins prior to the procedure. Immature oocytes released from small antral follicles were collected in connection with isolation of the cortex for OTC. The oocytes' maturation stage and the morphological characteristics of the cytoplasm, zona pellucida, perivitelline space and first polar body were assessed after in-vitro maturation (IVM)., Results: A total of 1649 immature oocytes were collected: 30% of oocytes matured to the metaphase II (MII) stage after IVM, while metaphase I (MI), germinal vesicle and degenerated oocytes accounted for 20%, 24% and 26%, respectively. The percentages of oocytes without any dysmorphisms were 53%, 92%, and 97% for the MII, MI and germinal vesicle stage oocytes, respectively. The most frequently observed dysmorphisms among the MII oocytes were first polar body fragmentation (22%), homogeneously distributed cytoplasmic granularity (16%) and an enlarged perivitelline space (14%). Interestingly, none of the oocytes at any stage had clusters of smooth endoplasmic reticulum (SER)., Conclusions: Morphological dysmorphisms are present among in-vitro-matured oocytes at all maturation stages. The incidence of dysmorphisms increases as maturation progresses. The most frequent dysmorphism among MII oocytes after IVM was fragmentation of the first polar body. Clusters of SER were not observed in oocytes from unstimulated patients., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Stress due to earthquakes and other natural catastrophic events may result in a decline in the male to female ratio of newborn infants. One reason may be an increased death of male fetuses 3-5 months earlier. Another reason may relate to reduced conception of males and/or early male embryo demise 8-10 months earlier. Almost all of the earthquakes and natural catastrophic events have caused declines in sex ratios at birth except the Hurricane Katrina which showed a rise in the birth sex ratio. We describe hypothetical immunological cause for the decline in the sex ratio at birth following periods of augmented stress levels., (Copyright © 2019. Published by Elsevier B.V.)

Background: Perfluoroalkyl substances (PFASs) are bio-accumulative pollutants, and prenatal exposure to PFASs is believed to impact human foetal development and may have long-term adverse health effects later in life. Additionally, maternal cigarette smoking may be associated with PFAS levels. Foetal exposure has previously been estimated from umbilical cord plasma, but the actual concentration in foetal organs has never been measured., Objectives: The concentrations of 5 PFASs and cotinine - the primary metabolite of nicotine - were measured in human foetuses, placentas, and maternal plasma to evaluate to what extent these compounds were transferred from mother to foetus and to determine if the PFAS concentrations were associated with maternal cigarette smoking., Methods: Thirty-nine Danish women who underwent legal termination of pregnancy before gestational week 12 were included; 24 maternal blood samples were obtained together with 34 placental samples and 108 foetal organs. PFASs and cotinine were assayed by liquid chromatography/triple quadrupole mass spectrometry., Results: In foetal organs, the average concentrations of perfluorooctanesulphonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDa), and perfluorodecanoic acid (PFDA) were 0.6ng/g, 0.2ng/g, 0.1ng/g, 0.1ng/g, and 0.1ng/g, respectively. A significant positive correlation was found between the exposure duration, defined as foetal age, and foetal to maternal ratio for all five PFASs and cotinine. Smokers presented 99ng/g cotinine in plasma, 108ng/g in placenta, and 61ng/g in foetal organs. No correlation between the maternal cotinine concentrations and PFAS concentrations was found., Conclusions: PFASs were transferred from mother to foetus, however, with different efficiencies. The concentrations of PFOS, PFOA, PFNA, PFUnDA, and PFDA in foetal organs were much lower than the maternal concentrations. Furthermore, a significant correlation between the exposure duration and all of the evaluated PFASs was found. The health-compromising concentrations of these substances during foetal development are unknown., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Life expectancy has increased by more than 30 years during the last century and continues to increase. Many women already live decades in menopause deprived of naturally produced oestradiol and progesterone, leading to an increasing incidence of menopause-related disorders such as osteoporosis, cardiovascular diseases and lack of general well-being. Exogenous oestradiol has traditionally been used to alleviate menopause-related effects. This commentary discusses a radical new method to postpone menopause. Part of the enormous surplus of ovarian follicles can now be cryostored in youth for use after menopause. Excision of ovarian tissue will advance menopause marginally and will not reduce natural fertility. Grafted tissue restores ovarian function with circulating concentrations of sex steroids for years in post-menopausal cancer survivors. Future developments may further utilize the enormous store of ovarian follicles. Currently, the main goal of ovarian cryopreservation is fertility preservation, but grafting of ovarian tissue may also serve endocrine functions as a physiological solution to prevent the massive medical legacy of osteoporosis and menopause-related conditions in the ageing population. This intriguing solution is now technically available; the question is whether this method qualifies for postponing menopause, perhaps initially for those patients who already have cryostored tissue?, (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Cochrane reviews are powerful tools, internationally recognized as the highest standard in evidence-based health care. A Cochrane analysis makes use of precise, reproducible criteria in the selection of studies for review. In the context of a previous Cochrane review (2010) on the subject of gonadotrophin-releasing hormone agonist (GnRHa) trigger, we questioned whether a review should be conducted during the research phase when new concepts are being developed. Recently, an updated Cochrane review was published, reaching the same general conclusion as the first one, i.e., GnRHa triggers lower the chance of pregnancy in fresh autologous IVF and intracytoplasmic injection treatment cycles. We argue that the new review repeats previous errors by compiling data from studies that were not comparable as different luteal phase protocols were used. From the clinical point of view, the luteal support used is the variable which affects the pregnancy rate and not the use of the GnRHa trigger for final oocyte maturation. Therefore, a meaningful comparison between GnRHa and HCG trigger must be confined to outcome measures that are not affected by the luteal support used. We conclude that the updated review falls short of addressing meaningful clinical and fundamental questions in the context of GnRHa trigger., (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Cryopreservation of ovarian tissue is one way of preserving fertility in young women with a malignant disease or other disorders that require gonadotoxic treatment. The purpose of the study was to explore how many women remained interested in continued cryostorage of their ovarian tissue beyond an initial 5-year period. Between 1999 and 2006, a total of 201 girls and young women had one ovary cryopreserved for fertility preservation in Denmark. One hundred of these met our inclusion criteria, which included a follow-up period of at least 5 years, and were mailed a questionnaire. The response rate was 95%. Sixteen of the patients (17%) stated that they wanted disposal of their tissue; the main reason was completion of family (63%). The mean age of those requesting disposal was 36.6 years, whereas those still wanting their tissue stored were significantly younger, with a mean age of 33.0 years (P < 0.008). In conclusion, most women with ovarian tissue cryobanked requested continued cryostorage after an initial period of at least 5 years. The main reason for requesting disposal was successful completion of a family., (Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the reproductive outcome in patients receiving frozen-thawed embryo transfer before and after doubling of the vaginal progesterone gel supplementation. The study was a retrospective study performed in The Fertility Clinic, Skive Regional Hospital, Denmark. A total of 346 infertility patients with oligoamenorrhoea undergoing frozen-thawed embryo transfer after priming with oestradiol and vaginal progesterone gel were included. The vaginal progesterone dose was changed from 90 mg (Crinone) once a day to twice a day and the reproductive outcome during the two periods was compared. The pregnancy rate increased significantly after doubling of the progesterone dose (26.7% (90 mg) versus 38.4% (180 mg); P=0.021). Moreover, the early pregnancy loss rate decreased significantly (67.4% versus 43.7%, respectively; P=0.014), which significantly increased the delivery rate (8.7% versus 20.5%, respectively; P=0.002). Doubling of the vaginal progesterone gel supplementation during frozen-thawed embryo transfer cycles decreased the early pregnancy loss rate, resulting in a significantly higher delivery rate. This study evaluated the reproductive outcome of 346 women with oligoamenorrhoea (cycle length >34 days) or amenorrhoea undergoing oestradiol and progesterone priming prior to frozen-thawed embryo transfer. Patients treated with vaginal progesterone gel (Crinone 90 mg) twice daily had a lower risk of pregnancy loss (43.7%) compared with women treated once a day (67.4%). This resulted in a significantly higher delivery rate (20.5% versus 8.7%, respectively)., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Some women suffering from leukemia require bone marrow transplantation to be cured. Bone marrow transplantation is associated with a high risk of sterility, and some patients are offered fertility preservation by cryopreservation of the ovarian cortex. Transplantation of the ovarian cortex to women cured of leukemia who became menopausal is currently not performed because of the risk of introducing the disease. In this study, individual pieces of ovarian cortex intended for reimplantation from 25 patients with leukemia were transplanted to each of 25 nude mice for 20 weeks. The ovarian cortex was examined before and after transplantation by histology and immunohistochemistry, and RT-quantitative PCR (in the 7 patients with a known marker). Seventeen patients had the ovarian cortex retrieved when they were in complete remission. Before transplantation, 4 of 7 pieces (2 from patients in complete remission) of ovarian cortex had a positive RT-quantitative PCR. After transplantation, none of the mice revealed any sign of disease, neither in the pieces of ovarian cortex transplanted nor in any of the murine organs evaluated. Thus, the ovaries from patients in complete remission do not appear to contain viable malignant cells contrasting ovarian tissue retrieved before treatment.

These three case reports describe the long-term duration of function of ovarian cortical tissue grafts among patients in a university fertility preservation programme in Europe and in a private practice programme in the USA. One woman underwent sterilizing cancer treatment and had frozen ovarian tissue transplanted, and two women underwent fresh ovarian tissue transplants. The function of ovarian cortical strips has continued for more than 7 years in these three women, with the birth of eight healthy babies following a single graft per patient. In addition to these three cases, transplantation (repeatedly in some cases) of cryopreserved ovarian tissue has restored reproductive function to all other women in the study centres' programmes for some years. The sustained longevity of function of the transplanted tissue suggests that it may also be possible to postpone the normal time of menopause or to alleviate its symptoms., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n=49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5h prior to freezing had previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility preservation; however, before applying the method clinically, each laboratory should perform thorough validation of their technique., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

A group of scientists from Harvard Medical School (Johnson et al., 2004) claims to have "established the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary," expressing no doubts about their methods, results and conclusion. Johnson et al. based their conclusions of oocyte and follicular renewal from existing germline stem cells (GSC) in the postnatal mouse ovary on three types of observations: (1) A claimed discordance in follicle loss versus follicle atresia in the neonatal period and in the following pubertal and adult period; (2) immunohistochemical detection of proliferating GSC with meiotic capacity using combined markers for meiosis, germline, and mitosis; and (3) neo-folliculogenesis in ovarian chimeric grafting experiments with adult mice. Oogenesis is the process that transforms the proliferative oogonium into an oocyte through meiosis, followed by folliculogenesis and follicular and oocyte maturation. The most crucial part in producing a functional oocyte is firstly, initiation and completion of the first meiotic prophase, and secondly, enclosure of the resulting diplotene oocyte in a follicle. Neither of these two events has been shown to take place in Johnson et al.'s study of the postnatal mouse ovary. We hereby address the observations underpinning their hypothesis and conclude that it is premature to replace the paradigm that adult mammalian neo-oogenesis/folliculogenesis does not take place.

The clinical efficacy of commercial gonadotrophin preparations has been the subject of an intense debate during recent years. Arguments have primarily focused on the origin of FSH activity (urine versus recombinant derived) and whether the preparation included LH-like activity. FSH isoform composition has received little or no attention, and is usually considered to have negligible effect on clinical effectiveness. By presenting the available data on the FSH isoform composition of commercial gonadotrophin preparations, the present paper challenges this assumption. To evaluate whether the FSH isoform composition affected the efficacy of a product, a meta-analysis was performed that compared a preparation expressing an acidic isoform profile (urinary-derived Metrodin-HP) with a preparation rich in less acidic isoforms (recombinant derived Gonal F). A total of five randomized clinical trials that specifically compared these two preparations was identified and included in the analysis. All parameters relating to the direct effect of FSH on the follicle differed significantly in favour of the product rich in less acidic isoforms, while data on pregnancy outcome did not reach significance. The importance of the FSH isoform profile and whether the FSH is derived from urine or by recombinant technique is discussed in relation to clinical efficacy. It is suggested that the FSH isoform profile of commercial gonadotrophin preparations is of clinical importance and should be taken into account when evaluating efficacy.

We assessed whether the smoking habits of parents around the time of conception affects the likelihood of the offspring being male or female. We found that the offspring sex ratio (male to female) was lower when either one or both of the parents smoked more than 20 cigarettes per day compared with couples in which neither of the parents smoked. We found the lowest sex ratio among children whose mothers and fathers both smoked more than 20 cigarettes per day (p<0.0001). Parental periconceptional smoking might be a contributing factor to a lower male to female sex ratio of offspring.