Functional Changes to Achilles Tendon and Enthesis in a Mouse Model of an Adolescent Masculine Gender-Affirming Hormone Treatment.

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis.

Addition of synthetic polymers to a conventional cryoprotectant solution in the vitrification of bovine ovarian tissue.

Some synthetic polymers can be used at low concentrations to reduce the toxicity of conventional cryoprotectant agents. In this study we investigated whether the addition of synthetic polymers to a conventional cryoprotectant solution would improve the cryopreservation of bovine ovarian tissue. Freshly collected ovaries from ten adult crossbred cows were incised using a scalpel in the frontal section. From each cow, ovarian cortical slices of 1 mm thickness were divided into 30 fragments of 3 × 3 mm, of which 10 served as fresh controls, 10 were vitrified with conventional cryoprotectant agents (2.93 M glycerol, 27 % w/v; 4.35 M ethylene glycol, 27 % w/v), and 10 were vitrified using the same cryoprotectant agents in addition to synthetic polymers (0.2 % PVP K-12, 0.2 % SuperCool X-1000 ™ w/v and 0.4 % SuperCool Z-1000 ™ w/v). After warming, histology was used to assess follicular quantity and integrity, while in vitro culture of mechanically isolated follicles encapsulated in an alginate matrix was performed for 15 days to assess their growth and hormonal production. Vitrified ovarian tissues presented abnormal morphology, a higher percentage of atretic follicles, and their isolated follicles had lower survival rates and lower frequency of antrum formation during in vitro culture compared to those from fresh tissue. At the end of culture, the follicles that had been cryopreserved produced less estradiol and progesterone than the fresh ones. The addition of synthetic polymers during tissue vitrification did not modify any of these parameters. We conclude that, under the conditions of this study, the use of this combination of synthetic polymers for tissue vitrification did not enhance the preservation of the morphological or functional integrity of bovine ovarian follicles.

Translocator protein expression and localization in human endometrium and endometriosis: A potential target for a noninvasive diagnosis?

The limitations of current imaging methods to detect small or superficial endometriotic lesions prompt the search for new molecular targets. TSPO is an 18 KDa protein located in the outer mitochondrial membrane, which can be traced by positron emission tomography (PET) using specific ligands. TSPO is located mostly in neurons and inflammatory sites outside the brain. We hypothesized that it might also be expressed in the human endometrium and endometrial-like tissue, being a target for molecular imaging of endometriosis. This prospective cross-sectional study included 28 women with endometriosis and 11 endometriosis-free controls. Endometriotic lesions (n = 49) and normal peritoneum (n = 13) from endometriosis patients were obtained during laparoscopy, while samples of eutopic endometrium from patients with endometriosis (n = 28) and from control women (n = 11) were collected in the operating room using a flexible device. TSPO mRNA expression was evaluated by quantitative reverse-transcription real-time PCR while protein expression was evaluated by immunohistochemistry with a monoclonal antibody antihuman TSPO. TSPO mRNA expression was detected in an invariable fashion in all tissue types evaluated; however, TSPO protein was found to be more abundant in the glandular epithelium than in the stroma, both in the endometrium and in the endometriotic lesions. Interestingly, hormone therapies did not alter the expression of TSPO, and its presence was mostly negative in tissues adjacent to endometriotic implants. As a proof of concept, the protein expression pattern of TSPO in endometriotic tissue and along the adjacent areas suggests that TSPO-based molecular imaging might be used for noninvasive endometriosis detection.

Bone quality following peripubertal growth in a mouse model of transmasculine gender-affirming hormone therapy.

During peri-puberty, bone growth and the attainment peak bone mass is driven predominantly by sex steroids. This is important when treating transgender and gender diverse youth, who have become increasingly present at pediatric clinics. Analogues of gonadotropin-releasing hormone (GnRH) are commonly prescribed to transgender and gender diverse youth prior to starting gender-affirming hormone therapy (GAHT). However, the impact of GnRH agonists on long bones with the addition of GAHT is relatively unknown. To explore this, we developed a trans-masculine model by introducing either GnRHa or vehicle treatment to female-born mice at a pre-pubertal age. This treatment was followed by male GAHT (testosterone, T) or control treatment three weeks later. Six weeks after T therapy, bone quality was compared between four treatment groups: Control (vehicle only), GnRHa-only, GnRHa + T, and T-only. Bone length/size, bone shape, mechanical properties, and trabecular morphology were modulated by GAHT. Independent of GnRHa administration, mice treated with T had shorter femurs, larger trabecular volume and increased trabecular number, higher trabecular bone mineral density, and wider superstructures on the surface of bone (e.g., third trochanters) when compared to control or GnRHa-only mice. In conclusion, prolonged treatment of GnRHa with subsequent GAHT treatment directly affect the composition, parameters, and morphology of the developing long bone. These findings provide insight to help guide clinical approaches to care for transgender and gender diverse youth.

Seminal glucose levels: a prognostic factor of sperm survival to cryopreservation?

OBJECTIVE: Considering that glucose is an important component of seminal plasma and is a cryoprotectant at high concentrations, the aim of this study was to investigate the possible association of glucose levels in fresh semen with the sperm survival and motility rates following cryopreservation. METHODS: This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analyzed according to the World Health Organization standards and glucose concentrations were measured using a dipstick glucometer. Samples were cryopreserved with Test Yolk Buffer-Gentamicine freezing medium under liquid nitrogen for an average of 120 days. The frozen aliquots were thawed at 37°C for 10 minutes and analyzed using the same methods and protocols used pre-freezing. RESULTS: Glucose levels ranged from 14 to 99 mg/dL and were similar in individuals with normal (n=100) vs. abnormal (n=49) semen analysis. The rates of sperm recovery (total, alive or motile sperm) in the cryopreserved samples did not change among samples with different glucose levels (p>0.05, Kruskal-Wallis ANOVA and Spearman's correlation coefficient). CONCLUSIONS: There appears to be no association between glucose levels in human semen samples and their resistance to cryopreservation.

A Mouse Model to Investigate the Impact of Gender Affirming Hormone Therapy with Estradiol on Reproduction.

Gender-affirming hormone therapy (GAHT) can help transgender and/or gender diverse (TGD) individuals achieve emobidment goals that align with their transition needs. Clinical evidence from estradiol (E)-GAHT patients indicate widespread changes in tissues sensitive to E and testosterone (T), particularly in the reproductive system. Notably, E-GAHTs effects on hormones and reproduction vary greatly between patients. With the goal of informing clinical research and practice for TGD individuals taking E, this study examines intact male mice implanted with capsules containing one of three different E doses (low 1.25 mg; mid 2.5 mg; high 5 mg), or a blank control capsule. All E-GAHT doses suppress T and follicle stimulating hormone levels while elevating E levels. Only the high E-GAHT dose significantly supresses luteinizing hormone levels. All E-GAHT doses affect epididymis tubule size similarly while seminiferous tubule morphology and bladder weight changes are dose-dependent. E-GAHT does not alter the presence of mature sperm, though E-exposed sperm have altered motility. These data represent the first evidence that mouse models offer an effective tool to understand E-GAHTs impact on reproductive health and the dose-dependent effects of this model permit examinations of diverse patient outcomes.

In vitro fertilization outcomes in a mouse model of gender-affirming hormone therapy in transmasculine youth.

OBJECTIVE: To investigate in vitro fertilization (IVF) outcomes in an adolescent transmasculine mouse model mimicking gender-affirming hormone therapy in prepubertal youth, both on testosterone (T) and after T washout. DESIGN: Experimental laboratory study using a validated mouse model. SETTING: University-based basic science research laboratory. ANIMAL(S): A total of 80 prepubertal 26-day-old C57BL/6N female mice were used in this study. INTERVENTION(S): Animals (n = 10/group) were implanted subcutaneously with gonadotropin-releasing hormone agonist at 3.6 mg or received sham surgery. After 21 days, they were implanted with silastic tubing containing either T 10 mg or placebo for 6 weeks. After 6 weeks, a group of animals were superovulated for immediate IVF, and another group had the implant removed and went through superovulation for IVF after 2 weeks (washout IVF). The total number of oocytes yielded, oocyte maturity rate, fertilization rate, and numbers of 2-cell embryos, 4-8-cell embryos, morula, blastocysts, and hatching blastocysts were recorded. RESULT(S): Testosterone treatment negatively impacted IVF outcomes in animals stimulated when receiving T, but not after T washout. Pretreatment with gonadotropin-releasing hormone agonist did not affect IVF outcomes. CONCLUSION(S): Although current T had a negative impact on IVF outcomes compared with controls, animals were still able to produce viable oocytes for fertilization and develop into blastocysts. Future efforts to study the impact of long-term T exposure on oocyte quality, especially aneuploidy rates, pregnancy outcomes, and live birth rates, are necessary.

Impaired in vitro fertilization outcomes following testosterone treatment improve with washout in a mouse model of gender-affirming hormone treatment.

BACKGROUND: The impact of gender-affirming testosterone on fertility is poorly understood, with ovarian histopathologic studies showing variable results, some with a detrimental effect on reproductive capacity and uncertain reversibility. Assisted reproductive outcome data are restricted to small case series that lack the ability to inform clinical practice guidelines and limit fertility preservation counseling for transgender and nonbinary individuals. OBJECTIVE: This study aimed to determine the impact of current testosterone and testosterone washout on in vitro fertilization outcomes in a mouse model for gender-affirming hormone treatment. We hypothesized that current or previous testosterone treatment would not affect in vitro fertilization outcomes. STUDY DESIGN: C57BL/6N female mice (n=120) were assigned to 4 treatment groups: (1) current control, (2) current testosterone, (3) control washout, and (4) testosterone washout. Testosterone implants remained in situ for 6 or 12 weeks, representing the short- and long-term treatment arms, respectively. Current treatment groups underwent ovarian stimulation with implants in place, and washout treatment groups were explanted and had ovarian stimulation after 2 weeks. Oocytes were collected, fertilized, and cultured in vitro, with one arm continuing to the blastocyst stage and the other having transfer of cleavage-stage embryos. Statistical analysis was performed using GraphPad Prism, version 9.0 and R statistical software, version 4.1.2, with statistical significance defined by P<.05. RESULTS: Current long-term testosterone treatment impaired in vitro fertilization outcomes, with fewer mature oocytes retrieved (13.7±5.1 [standard deviation] vs 28.6±7.8 [standard deviation]; P<.0001) leading to fewer cleavage-stage embryos (12.1±5.1 vs 26.5±8.2; P<.0001) and blastocysts (10.0±3.2 vs 25.0±6.5; P<.0001). There was recovery of in vitro fertilization outcomes following washout in the short-term treatment cohort, with incomplete reversibility in the long-term cohort. Testosterone did not negatively affect maturity, fertilization, or blastulation rates. CONCLUSION: In a mouse model of gender-affirming hormone treatment, testosterone negatively affected oocyte yield without affecting oocyte quality. Our findings suggest that testosterone reversibility is duration-dependent. These results demonstrate the feasibility of in vitro fertilization without testosterone discontinuation while supporting a washout period for optimization of mature oocyte yield.

Presence of ovarian stromal aberrations after cessation of testosterone therapy in a transgender mouse model†.

Some transmasculine individuals may be interested in pausing gender-affirming testosterone therapy and carrying a pregnancy. The ovarian impact of taking and pausing testosterone is not completely understood. The objective of this study was to utilize a mouse model mimicking transmasculine testosterone therapy to characterize the ovarian dynamics following testosterone cessation. We injected postpubertal 9-10-week-old female C57BL/6N mice once weekly with 0.9 mg of testosterone enanthate or a vehicle control for 6 weeks. All testosterone-treated mice stopped cycling and demonstrated persistent diestrus within 1 week of starting testosterone, while control mice cycled regularly. After 6 weeks of testosterone therapy, one group of testosterone-treated mice and age-matched vehicle-treated diestrus controls were sacrificed. Another group of testosterone-treated mice were maintained after stopping testosterone therapy and were sacrificed in diestrus four cycles after the resumption of cyclicity along with age-matched vehicle-treated controls. Ovarian histological analysis revealed stromal changes with clusters of large round cells in the post testosterone group as compared to both age-matched controls and mice at 6 weeks on testosterone. These clusters exhibited periodic acid-Schiff staining, which has been previously reported in multinucleated macrophages in aging mouse ovaries. Notably, many of these cells also demonstrated positive staining for macrophage markers CD68 and CD11b. Ovarian ribonucleic acid-sequencing found upregulation of immune pathways post testosterone as compared to age-matched controls and ovaries at 6 weeks on testosterone. Although functional significance remains unknown, further attention to the ovarian stroma may be relevant for transmasculine people interested in pausing testosterone to carry a pregnancy.

A mouse model mimicking gender-affirming treatment with pubertal suppression followed by testosterone in transmasculine youth.

STUDY QUESTION: Can mice serve as a translational model to examine the reproductive consequences of pubertal suppression with GnRH agonist (GnRHa) followed by testosterone (T) administration, a typical therapy in peripubertal transmasculine youth? SUMMARY ANSWER: An implanted depot with 3.6 mg of GnRHa followed by T enanthate at 0.45 mg weekly can be used in peripubertal female mice for investigating the impact of gender-affirming hormone therapy in transmasculine youth. WHAT IS KNOWN ALREADY: There is limited knowledge available in transgender medicine to provide evidence-based fertility care, with the current guidelines being based on the assumption of fertility loss. We recently successfully developed a mouse model to investigate the reproductive consequences of T therapy given to transgender men. On the other hand, to our knowledge, there is no mouse model to assess the reproductive outcomes in peripubertal transmasculine youth. STUDY DESIGN, SIZE, DURATION: A total of 80 C57BL/6N female mice were used in this study, with n = 7 mice in each experimental group. PARTICIPANTS/MATERIALS, SETTING, METHODS: We first assessed the effectiveness of GnRHa in arresting pubertal development in the female mice. In this experiment, 26-day-old female mice were subcutaneously implanted with a GnRHa (3.6 mg) depot. Controls underwent a sham surgery. Animals were euthanized at 3, 9, 21 and 28 days after the day of surgery. In the second experiment, we induced a transmasculine youth mouse model. C57BL/6N female mice were subcutaneously implanted with a 3.6 mg GnRHa depot on postnatal day 26 for 21 days and this was followed by weekly injections of 0.45 mg T enanthate for 6 weeks. The control for the GnRH treatment was sham surgery and the control for T treatment was sesame oil vehicle injections. Animals were sacrificed 0.5 weeks after the last injection. The data collected included the day of the vaginal opening and first estrus, daily vaginal cytology, weekly and terminal reproductive hormones levels, body/organ weights, ovarian follicular distribution and corpora lutea (CL) counts. MAIN RESULTS AND THE ROLE OF CHANCE: GnRHa implanted animals remained in persistent diestrus and had reduced levels of FSH (P = 0.0013), LH (P = 0.0082) and estradiol (P = 0.0155), decreased uterine (P < 0.0001) and ovarian weights (P = 0.0002), and a lack of CL at 21 days after GnRHa implantation. T-only and GnRHa+T-treated animals were acyclic throughout the treatment period, had sustained elevated levels of T, suppressed LH levels (P < 0.0001), and an absence of CL compared to controls (P < 0.0001). Paired ovarian weights were reduced in the T-only and GnRHa+T groups compared with the control and GnRHa-only groups. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although it is an appropriate tool to provide relevant findings, precaution is needed to extrapolate mouse model results to mirror human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this study describes the first mouse model mimicking gender-affirming hormone therapy in peripubertal transmasculine youth. This model provides a tool for researchers studying the effects of GnRHa-T therapy on other aspects of reproduction, other organ systems and transgenerational effects. The model is supported by GnRHa suppressing puberty and maintaining acyclicity during T treatment, lower LH levels and absence of CL. The results also suggest GnRHa+T therapy in peripubertal female mice does not affect ovarian reserve, since the number of primordial follicles was not affected by treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Michigan Institute for Clinical and Health Research grants KL2 TR 002241 and UL1 TR 002240 (C.D.C.); National Institutes of Health grants F30-HD100163 and T32-HD079342 (H.M.K.); University of Michigan Office of Research funding U058227 (A.S.); American Society for Reproductive Medicine/Society for Reproductive Endocrinology and Infertility grant (M.B.M.); and National Institutes of Health R01-HD098233 (M.B.M.). The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core Facility was supported by the Eunice Kennedy Shriver NICHD/NIH grants P50-HD028934 and R24-HD102061. The authors declare that they have no competing interests.

Altered Endometrial Expression of α-Inhibin Subunit and Its Co-Receptor Betaglycan in Infertile Women with Endometriosis.

BACKGROUND: Inhibins and their co-receptor betaglycan are members of the transforming growth factor ß superfamily, a group of signaling molecules that control the differentiation of human endometrium in the secretory phase of the menstrual cycle. OBJECTIVE: Since endometriosis is associated with endometrial dysfunction and infertility, this study aimed at evaluating the expression of α-inhibin and betaglycan mRNA and proteins in endometrial samples of infertile women with and without endometriosis. DESIGN: This was a cross-sectional study. Participants/Materials: Endometrial samples of women with (n = 17) and without (n = 22) endometriosis were subdivided according to the menstrual cycle phase into proliferative and secretory. SETTING: University hospital. METHODS: We used real-time RT-PCR to quantify mRNA levels and immunohistochemistry to localize the proteins. RESULTS: α-inhibin mRNA levels were significantly increased in the secretory phase (p < 0.01 vs. proliferative phase) only among women with endometriosis. Conversely, betaglycan mRNA levels were downregulated in the secretory endometrium of controls (p < 0.01 vs. proliferative) but failed to change between cycle phases of patients with endometriosis. Both proteins were present in the glandular epithelium and stroma in the endometrium of women with and without endometriosis. Immunostaining analysis showed that while α-inhibin protein expression did not vary significantly, the intensity of betaglycan immunostaining decreased in the secretory phase in the control group (p = 0.038 vs. proliferative phase) but not in the endometriosis group. LIMITATIONS: We cannot determine whether endometriosis causes the abnormal expression of α-inhibin and betaglycan in the eutopic endometrium or if this alteration already existed before the establishment of endometriotic lesions. CONCLUSION: Our findings suggest an abnormally increased expression of α-inhibin mRNA (not protein) and betaglycan (mRNA and protein) in the secretory-phase endometrium of women with endometriosis.

Pharmacokinetic comparison of three delivery systems for subcutaneous testosterone administration in female mice.

Transmasculine individuals are often prescribed testosterone (T) for masculinizing hormone therapy. Mouse models mimicking transmasculine T therapy require reliable long-term T administration. The objectives of this study were three-fold, namely, to compare: 1) the release dynamics of three different subcutaneous delivery systems of T enanthate administration (subcutaneous injections, commercially available pellets, and silastic implants) over a 6-week period in postpubertal C57BL/6N mice, 2) to compare the timing for T levels in plasma to return to baseline and cyclicity to resume after cessation of T between injections and pellets, 3) to utilize silastic implants to achieve sustainable increase in T levels utilizing T enanthate and crystalline T. All three modes of T administration resulted in an increase in T levels in plasma. Pharmacokinetic analyses showed a similar overall exposure to T enanthate over 6 weeks (integrated area) for, subcutaneous injection (0.45 mg two times per week and 0.90 mg one time per week), pellet (5 mg 60-day release), and silastic implant (5 mg 21 week) groups. Crystalline T had lower solubility and a decreased integrated area compared to T enanthate, even when implanted at a higher dosage, indicating different pharmacokinetic profiles based on type of T formulation when utilizing the same silastic delivery method. Surgical removal of pellets and silastic tubing led to a quick drop in T levels and resumption of estrous cyclicity, while cessation of injections required a long washout period for T levels to drop and estrous cycles to resume. Sustained elevation in T levels was achieved for at least 21 weeks with silastic implants. As all three delivery methods are able to elevate T levels in female mice for at least 6 weeks, choice of T administration method should be based on outcomes of interest and study design.

Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

BACKGROUND: Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. OBJECTIVE AND DESIGN: In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. RESULTS: The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

Reversibility of testosterone-induced acyclicity after testosterone cessation in a transgender mouse model.

OBJECTIVE: To establish if the cessation of testosterone (T) therapy reverses T-induced acyclicity in a transgender mouse model that allows for well-defined T cessation timing. DESIGN: Experimental laboratory study using a mouse model. SETTING: University-based basic science research laboratory. ANIMALS: A total of 10 C57BL/6NHsd female mice were used in this study. INTERVENTION(S): Postpubertal C57BL/6NHsd female mice were subcutaneously implanted with T enanthate (n = 5 mice) or placebo (n = 5 mice) pellets. Pellets were surgically removed after 6 weeks to ensure T cessation, after which the mice were followed for four estrous cycles after the resumption of cyclicity. MAIN OUTCOME MEASURE(S): Primary outcomes included daily vaginal cytology and weekly T levels before, during, and after T enanthate or placebo pellet implantation and removal. Secondary outcomes included ovarian follicle distribution and corpora lutea numbers, body metrics, and terminal diestrus hormone levels. RESULT(S): T-treated mice (100%) resumed cycling within one week of T pellet removal after six weeks of T therapy. T levels were significantly elevated during T therapy and decreased to control levels after surgical pellet removal. No detectable differences were observed in the follicle count, corpora lutea formation, diestrus hormone levels, or body metrics after four estrous cycles, with the exception of persistent increased clitoral area between T-treated mice and controls. One T-treated mouse was sacrificed early due to vaginal prolapse and not included in subsequent analyses. CONCLUSION(S): Our results demonstrated a close temporal relationship between estrous cycle return and T levels dropping to control levels following T pellet removal. The return of regular cyclic ovulatory function is also supported by the formation of corpora lutea and the lack of detectable differences in key reproductive parameters as compared to controls four cycles after T cessation. These results may be relevant to understanding the reversibility of T-induced amenorrhea and possible anovulation in transgender men interested in pausing T to pursue pregnancy or oocyte donation. Results may be limited by the duration of T treatment, lack of functional testing, and physiological differences between mice and humans.

Seminal protein levels assessed with dipstick test correlate with sperm recovery after cryopreservation.

The process of freezing/thawing causes structural and functional damage to sperm samples, which can be mitigated by seminal plasma proteins. This study investigated the proposition that seminal protein measurements using a urinary dipstick prior to freezing could help predict the post-thaw recovery of live spermatozoa. This was a prospective study including 149 men undergoing semen analysis due to male and/or female infertility. The seminal samples were analysed according to World Health Organisation standards and protein concentrations were measured using a commercially available urinary dipstick following quantitative validation. The median live sperm recovery rates were 79%, 81% and 94%, respectively, in samples with protein concentrations of ≤1.0 g/L (+/++), 3.0 g/L (+++) and ≥20.0 g/L (++++) measured in fresh specimen dipstick analysis (p < 0.05) indicating that the probability of recovering at least 50% of frozen spermatozoa increased progressively with higher protein concentrations in the fresh sample (chi-square for linear association = 7.17. p = 0.007). In conclusion, fresh seminal protein concentration levels assessed with a dipstick test correlate with the proportion of live spermatozoa recovered from cryopreserved samples. This simple, low-cost test may add prognostic information to baseline semen analysis prior to sperm banking.

ADVERSE EVENTS OF THE ARGUS II RETINAL PROSTHESIS: Incidence, Causes, and Best Practices for Managing and Preventing Conjunctival Erosion.

PURPOSE: To analyze and provide an overview of the incidence, management, and prevention of conjunctival erosion in Argus II clinical trial subjects and postapproval patients. METHODS: This retrospective analysis followed the results of 274 patients treated with the Argus II Retinal Prosthesis System between June 2007 and November 2017, including 30 subjects from the US and European clinical trials, and 244 patients in the postapproval phase. Results were gathered for incidence of a serious adverse event, incidence of conjunctival erosion, occurrence sites, rates of erosion, and erosion timing. RESULTS: Overall, 60% of subjects in the clinical trial subjects versus 83% of patients in the postapproval phase did not experience device- or surgery-related serious adverse events. In the postapproval phase, conjunctival erosion had an incidence rate of 6.2% over 5 years and 11 months. In 55% of conjunctival erosion cases, erosion occurred in the inferotemporal quadrant, 25% in the superotemporal quadrant, and 20% in both. Sixty percent of the erosion events occurred in the first 15 months after implantation, and 85% within the first 2.5 years. CONCLUSION: Reducing occurrence of conjunctival erosion in patients with the Argus II Retinal Prosthesis requires identification and minimization of risk factors before and during implantation. Implementing inverted sutures at the implant tabs, use of graft material at these locations as well as Mersilene rather than nylon sutures, and accurate Tenon's and conjunctiva closure are recommended for consideration in all patients.

Conditional Fgfr1 Deletion in GnRH Neurons Leads to Minor Disruptions in the Reproductive Axis of Male and Female Mice.

In humans and mice, inactivating mutations in fibroblast growth factor receptor 1 (Fgfr1) lead to gonadotropin-releasing hormone (GnRH) deficiency and a host of downstream reproductive disorders. It was unclear if Fgfr1 signaling directly upon GnRH neurons critically drove the establishment of a functional GnRH system. To answer this question, we generated a mouse model with a conditional deletion of Fgfr1 in GnRH neurons using the Cre/loxP approach. These mice, called Fgfr1cKO mice, were examined along with control mice for their pubertal onset and a host of reproductive axis functions. Our results showed that Fgfr1cKO mice harbored no detectable defects in the GnRH system and pubertal onset, suffered only subtle changes in the pituitary function, but exhibited significantly disrupted testicular and ovarian morphology at 25 days of age, indicating impaired gametogenesis at a young age. However, these disruptions were transient and became undetectable in older mice. Our results suggest that Fgfr1 signaling directly on GnRH neurons supports, to some extent, the reproductive axis function in the period leading to the early phase of puberty, but is not critically required for pubertal onset or reproductive maintenance in sexually mature animals.

C-type natriuretic peptide signaling in human follicular environment and its relation with oocyte maturation.

Studies in mice have shown that C-type natriuretic peptide (CNP) is produced by granulosa cells and contributes to ovarian follicle growth and oocyte meiotic arrest until the preovulatory LH surge. In humans, the relationship between intraovarian CNP levels and oocyte meiotic resumption is unknown. The aim of this study was to investigate whether CNP and its receptor NPR2 are expressed in human ovarian follicles and if their levels change according to the meiotic phase of oocytes. We collected follicular fluid (FF) and luteinized granulosa cells (LGC) from follicle pools (n = 47), and FF, LGC and cumulus cells (CC) from individual follicles (n = 96) during oocyte pickup for in vitro fertilization. There was a positive linear correlation between CNP levels in FF pools and basal antral follicle counting (rs = 0.458; p = 0.002), number of preovulatory follicles >16 mm (rs = 0.361; p = 0.016) and number of oocytes retrieved (rs = 0,378; p = 0.011) and a negative correlation between CNP levels in FF pools and the percentage of mature (MII) oocytes retrieved (rs = -0.39; p = 0.033). FF CNP levels in follicles containing MII oocytes were significantly lower than in follicles containing immature (MI) oocytes (median = 0.44 vs. 0.57 ng/mL, p < 0.05). Accordingly, the CNP precursor gene NPPC was 50% less expressed in LGC from follicles containing MII oocytes than in follicles containing MI oocytes (p < 0.01). In addition, NPR2 mRNA was down-regulated in CC surrounding MII oocytes (60% reduction, p < 0.01). CNP signaling is downregulated in human ovarian follicles containing mature oocytes. Further studies should clarify whether CNP signaling is essential to keep oocyte meiotic arrest in humans.

Caspase-3 gene expression in human luteinized granulosa cells is inversely correlated with the number of oocytes retrieved after controlled ovarian stimulation.

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman's r = -0.308), the number of collected oocytes (r = -0.451), the number of mature oocytes (r = -0.526), the number of fertilized oocytes (r = -0.439) and the number of viable embryos (r = -0.443, all statistically significant at p < 0.02 level). No such associations were found with caspase-8, bax or bcl-2. These preliminary findings suggest that increased caspase-3 gene expression in granulosa cells is associated with a worse ovulatory response in humans.

Mindfulness-based program for stress reduction in infertile women: Randomized controlled trial.

Infertile women often experience chronic stress, which may have a negative impact on general well-being and may increase the burden of infertility. In this open-label, parallel, randomized controlled trial, infertile women aged 18-50 years (median 37 years) were assigned to an 8-week mindfulness-based program (MBP) or no intervention. The primary outcome was stress severity measured by the Lipp's Stress Symptoms Inventory (ISSL). Data were analyzed by modified intent-to-treat principle, which included all cases available to follow-up regardless of adherence to the intervention (62 participants from the MBP group and 37 from the control group). The median number of symptoms of chronic stress recorded in the past month decreased from six (interquartile range 2 to 9) before the MBP to two (interquartile range 1 to 4) after the intervention (p < 0.001, repeated measures analysis of variance with Time × Group interaction). Depressive symptoms also decreased after MBP, whereas general well-being improved (p < 0.01 for both outcomes). Hair cortisol and serum brain-derived neurotrophic factor (BDNF) did not change significantly between preintervention and postintervention. None of the outcomes changed significantly in the control group. MBP was effective in reducing stress and depressive symptoms while increasing general well-being in infertile women.