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Clinical aspects of histological and hormonal parameters in boys with cryptorchidism

2022; Wiley; Volume: 130; Issue: S143 Linguagem: Inglês

10.1111/apm.13247

1600-0463

Simone Hildorf,

Urological Disorders and Treatments

Principal supervisor: Professor Dina Cortes, MD, DMSc Department of Clinical Medicine, University of Copenhagen, Denmark Department of Pediatrics and Adolescent Medicine, Copenhagen University Hospital Hvidovre, Denmark Co-supervisors: Professor Magdalena Elisabeth Fossum, MD, PhD Department of Clinical Medicine, University of Copenhagen, Denmark Department of Pediatric Surgery, Copenhagen University Hospital Rigshospitalet, Denmark Chief consultant Erik Clasen-Linde, MD Department of Pathology, Copenhagen University Hospital Rigshospitalet, Denmark Chairman: Professor Ulla Nordström Joensen, MD, PhD Department of Clinical Medicine, University of Copenhagen, Denmark Department of Urology, Copenhagen University Hospital Rigshospitalet, Denmark Assessment committee: Professor Aleksander Giwercman, MD, DMSc Department of Translational Medicine, Clinical Research Centre, University of Lund, Sweden Reproductive Medicine Centre, Skåne University Hospital, Malmö, Sweden Professor Christian Radmayr, MD, PhD Department of Pediatric Urology, Medical University Innsbruck, Austria All patient photos in the thesis are published after written consent from the parents. All figures and illustrations have been created with BioRender.com by Simone Engmann Hildorf. Publication license is authorized. This project received financial support from the Novo Nordisk Foundation (grant no. NNF SA170030576), the Queen Louise's Children's Hospital Foundation, the Foundation of Christian and Ottilia Brorson, Julie von Müllens Foundation, the Graduate School of Health and Medical Sciences, and the European Reference Network for rare diseases. To Aleksander PAPERS INCLUDED IN THIS THESIS A clinical cohort study with retrospective assessment of testicular biopsies and reproductive hormonal values. Studied: 333 boys aged 34–390 (median age 274) days. 69 (21%) boys had bilateral cryptorchidism. 25% had reduced G/T, 23% lacked Ad spermatogonia, and two boys had no germ cells. Inhibin B significantly correlated with G/T and AdS/T, a total of 70 boys (21%) had inhibin B below 2.5th percentile. About 20% to 25% of boys with nonsyndromic cryptorchidism possibly have a risk for infertility despite early orchiopexy during the first year of life. as Paper I Studied: 67 boys aged 2–7.0 (3.8 years) years diagnosed with bilateral ascended testes compared to 86 boys median aged 3.9 years (2–6.9 years) with late referral bilateral congenital cryptorchidism The fertility potential was equally impaired in boys with bilateral ascending testes and bilateral congenital cryptorchidism. Ascended testes should be surgically corrected when the diagnosis is settled. as Paper I and a clinical follow-up study. Studied: 208 boys with bilateral cryptorchidism aged 4 months to 9 (1.7) years. The median age at follow-up was 2.7 years. Inhibin B MoM improved significantly from time of surgery to follow-up. Orchidopexy before 1 year of age expressed the most favourable improvement, as inhibin B MoM significantly increased for this age group (p < 0.03). Totally, at 1-year follow-up, inhibin B was below 2.5th percentile in 26% of the boys. Surgery does matter. At time of follow-up, 26% of these boys with surgery for bilateral cryptorchidism may risk infertility. Some boys could be suspected of an endocrinopathy. as Paper I Studied: 35 boys aged 37–159 (124) days. Five (14%) boys had bilateral cryptorchidism. Nine (26%) had G/T below lower range. Two of them could be expected of an endocrinopathy as FSH were below or just above 2.5th percentile. 97% had normal male LH/FSH ratio. 54% (19/35, 95% CI 0.37–0.71) had normal G/T and AdS/T as well as all hormones within normal range. The majority of boys with congenital nonsyndromic cryptorchidism exhibit normal minipuberty pattern. Few cases could be suspected of an endocrinpathy. Do parents accept experimental cryopreservation? – a pioneer study. Is the rate of acceptance different when offered as an additional procedure vs as part of bilateral orchiopexy? Offering cryopreservation to parents as an experimental fertility preservation option for their prepubertal boy. Studied: 14 parents of boys with severely reduced fertility potential who were offered cryopreservation as an additional procedure vs 27 parents of boys at time of bilateral orchidopexy. The acceptance rate to perform testicular tissue cryopreservation was 90% (95% CI 0.77–0.97) with 37 out of 41 parents. No difference between in the two groups 93% (13/14) vs 89% (24/27, p = 0.68). Study Design Fiala et al. (2021) Prospective randomized 36 unilateral (21 vs 15) 2.5–3.5 months at inclusion. 6 months at drug treatment start and orchidopexy within 12 months Gonadorelin (Kryptocur®) i.n. 0.2 mg (0.1 mL) x 6 every day for 4 weeks Pre-orchidopexy Concentrations of LH, FSH, testosterone, inhibin B, AMH, penile size and testicular size were evaluated at 3 months of age and at orchidopexy Thorup et al. (2018) Prospective case–control 10 bilateral (5 vs 5) 0.6–3.5 years at orchidopexy. Drug treatment started 3 months later Cryopreservation and second biopsy 12 months later Post-orchidopexy Histology before and after: G/T (at least 100) and Ad spermatogonia per cross-sectional tubule (at least 250). Offered cryopreservation 12 months after primary orchidopexy No side effects None of boys with G/T less than 0.2 improvedsignificantly, whether they were treated or not Vincel et al. (2018) Prospective randomized 10 bilateral (5 vs 5) 7–55 (median 20 vs 22 between the two groups) months (p = 0.32) at orchidopexy. Drug treatment immediately after 10–62 (median 27 vs 30) months (p = 0.18) at second biopsy Buserelin (Suprefact®) i.n. 10 μg every second day in 6 months Post-orchidopexy Histology before and after: G/T (at least 100) and Ad spermatogonia per cross-sectional tubules (at least 100) At surgery, G/T was similar (p = 0.67) Improved fertility potential as G/T increased within the treated group: median 0.11 vs 0.43 (p = 0.03) compared to no change found in the surgery alone group Ad spermatogonia only appeared after treatment (all lacked at inclusion) No side effects Unknown and none-authorized dilution of buserelin. Jallouli et al. (2009) Prospective randomized 24 unilateral (12 vs 12) Pre-orchidopexy Histology: Ad spermatogonia per tubule (at least 50) Improved fertility potential as mean Ad spermatogonia per tubule was greater: 0.88 vs 0.49 (p = 0.002) Only significance in boys treated after 3 years Zivkovic et al. (2009) Prospective case–control 55 unilateral (32 vs 33) 17/32: Buserelin (Suprefact®) i.n. 20 μg every day for 28 days + hCG i.m. 1500 IU every week for 3 weeks 15/32: received hCG i.m. 1500 IU every week for 3 weeks Pre-orchidopexy Histology: Ad spermatogonia per tubule (at least 50) Testosterone: Before, 14 days after Buserelin®, 24 h after each hCG, and 3 months after orchidopexy Improved fertility potential as more treated boys had number of Ad spermatogonia per tubules above 0.1 with 53% vs 18% (p = 0.019) Those with normal Ad spermatogonia count had better sufficient Leydig cell function by testosterone concentration (p < 0.003) No side effects None-authorized dilution of buserelin. Schwentner et al. (2005) Prospective randomized 42 unilateral and bilateral (21:12 unilateral vs 21:9 unilateral) Gonadorelin i.n. 1.2 mg every day for 4 weeks before orchidopexy Pre-orchidopexy Histology: Ad spermatogonia per tubule (at least 80) Improved fertility potential as mean Ad spermatogonia per tubule was greater: 1.05 vs 0.52 (p = 0.007) Treated boys had 101.9% more spermatogonia per tubule Best effect in bilateral cases (0.96 vs 0.56, p = 0.005) No side effects Best effect within the first year Reduced G/T; below lower range Reduced AdS/T; below 0.01 Jørgen Thorup, my mentor—you are an endless source of wisdom and a wonderful teacher, to have completed a PhD under your guidance is a privilege I will never forget. I am grateful for all our trips around the world and for you challenging my knowledge in everything from fetal germ cell development to spermatogenesis and the minipuberty in mice to the greatest classical tunes of Mozart. Thank you so much! To Dina Cortes, my principal supervisor. Thank you for being the most brilliant supervisor and for supporting me in both private and professional matters. Without your assistance and dedicated involvement in every single step throughout the process, this project would have never been accomplished. I am deeply thankful for everything you have taught me and look forward to continuing with our next papers. To my co-supervisor, Magdalena Fossum, I want to express my gratitude for giving me the opportunity as this project would not have been possible without the grant from Novo Nordisk Foundation. Your work speaks volumes of the kind of power woman you are—efficient, organized, result-oriented, and well-liked. It is a great pleasure to work under such an enthusiastic and inspiring person. I wish to express my sincere thanks to my co-supervisor Erik Clasen-Linde for taking me into the Department of Pathology and teaching me the importance and beauty of pathology. I have appreciated the freedom you have given me in my work and the support you have provided, whenever I have asked for it. Susanne Reinhardt, an amazing pediatric surgeon who directly believed in me and introduced me to pediatric surgery. Thank you for encouraging me to talk to Jørgen and Dina for a research project during medical school! Many hugs to the surgical pediatric team: Jacob, Kolja, Peter, Gert, Lars, and Inge. I have always felt welcomed and enjoyed our conversations. I am also grateful to John Hutson for giving me the opportunity to visit the Royal Children's Hospital in Melbourne. Thank you Ruili, Gulcan, and Jaya for taking so good care of me and not only making the time in your laboratory a blast but also in your amazing city. A big thanks to my fellow pathology PhD students Ingvild, Ausrine, Lauge, Charlotte, Andrea, Nabi, and Marina for your endless support, advice, humor, and for putting up with me throughout the years. And dearest Simon, how we all miss you! To Camilla and Niels for help in the laboratory and in the scanning room, and for generally making the Department of Pathology a fun place to work. A warm thank you to the Tissue Engineering family; Clara, Oliver, Nikolaj, Fatemeh, and Carmen for your engagement and support. You guys convert unsolvable problems into exciting challenges. Jens Hillingsøe, head of the Department of Surgical Gastroenterology, and Jane Preuss Hasselby, head of the Department of Pathology, are thanked for their support and for letting me use the facilities. I am very fortunate to be a member of the Fertility Restoration Consortium and CAG-SURF. Thank you all for motivating me to do more research and for extraordinary good scientific sparring. A special thanks to Claus Yding Andersen and the rest of the staff at the Laboratory of Reproductive Biology for always welcoming me with smiles and hugs, and to Murat, Lihua, and Danyang for excellent humor and music. Finally, I owe my deepest gratitude to Aleksander, my fiancé and the love of my life. I am forever thankful for the unconditional love and support every day. My appreciation also goes out to my lovely family (my mum, dad, sister, brother, and the Enoch family) and friends for their encouragement and support all through my studies. Thank you, Andrea—I look forward to reading your own thesis one day. Ad spermatogonia, Type A dark spermatogonia; AdS/T, Number of Ad spermatogonia per cross-sectional seminiferous tubule; AMH, Anti-Müllerian hormone; CD99, Cluster of differentiation 99 (also referred to as MIC-2); CI, Confidence interval; c-KIT, KIT proto-oncogene; D2-40, Anti-podoplanin M2A antigen (also referred to as M2A); DAZL, Deleted in azoospermia like; DC, Dina Cortes, MD, DMSc.; ECL, Erik Clasen-Linde, MD.; FSH, Follicle-stimulating hormone; G/T, Number of germ cells (gonocytes, spermatogonia, and spermatocytes) per cross-sectional seminiferous tubule; GnRH, Gonadotropin-releasing hormone; hCG, Human chorionic gonadotropin; HE, Hematoxylin and eosin; HPA, Hypothalamic–pituitary gonadal axis; INSL3, Insulin-like 3 hormone; JT, Jørgen Thorup, MD, PhD.; KK, Kolja Peter Kvist, MD.; LH, Luteinizing hormone; LIN28, Lin-28 homolog A; MAGE-A4, Melanoma-associated antigen A4; MoM, Multiple of the median; NANOG, Homeobox protein Nanog; Oct3/4, Octamer-binding transcription factor 3/4 (also known as POU5F1); PAS, Periodic acid-Schiff; PGC, Primordial germ cells; PLAP, Placental alkaline phosphatase; RXFP2, Relaxin/insulin-like family peptide receptor 2; SC/T, Sertoli cell number per cross-sectional tubules SOX9: SRY-box transcription factor 9; SH, Simone Hildorf, MD, PhD student; SRY, Sex-determining region Y; SSC, Spermatogonial stem cell; UTF-1, Undifferentiated embryonic cell transcription factor 1; VASA, Dead-box helicase 4 (also known as DDX4). Cryptorchidism (from the Greek "hidden testis") is the failure of descent of one or both testes to the scrotum [1, 2]. In the western world, about 2.5% of all boys undergo surgery for cryptorchidism, thus it remains one of the most common surgical procedures performed in boys [3, 5, 4]. In general, cryptorchidism is considered a mild anomaly, but it can have serious effects on men's health in adulthood as it represents the best-characterized risk factor for infertility and testis cancer. Men with a history of cryptorchidism make up around 20%–27% of azoospermic men [6, 7]. Men with former bilateral cryptorchidism have significantly lower paternity rates (65%) in comparison to men with former unilateral cryptorchidism (90%) and control men (93%) [8, 9]. In addition, such patients have a greater risk of testicular neoplasia than the general population [10-15]. Nowadays, early surgical correction is the gold standard for cryptorchidism with the prospects of minimizing germ cell loss and damage. The Nordic consensus on the treatment of cryptorchidism advocates for surgery starting at 6 months and preferably performed by 12 months of age [16]. The European Association of Pediatric Urology recommends orchidopexy from 6 months and within the subsequent year, by 18 months at the latest [17, 18], whereas the American Urological Association advises orchidopexy between 6 and 18 months of age [2]. However, the effect of early orchidopexy has sparsely been evaluated, and surgery in childhood does not guarantee subsequent fertility and paternity [19-22]. Consequently, an important issue is how to identify prepubertal boys with cryptorchidism at high risk of infertility at surgery and monitor testicular function after surgical treatment. Moreover, it is a challenge to identify and treat boys who develop cryptorchidism after birth. The intention of this thesis is to increase knowledge of the fertility potential in boys with cryptorchidism. Testicular histology with pathohistological grading together with the status of reproductive hormones obtained at the surgery are markers of importance for future fertility (the fertility potential), whereas the status of reproductive hormones can be followed to monitor testicular function after treatment. This unique data can help clinicians identify those patients with a high risk of infertility, which is the first step to potentially changing our approach and management. The main treatment goals for boys with cryptorchidism are to give them the ability to father biological children and avoid testicular cancer. However, this might not be achievable by early surgical intervention alone. If there is an underlying endocrinopathy resulting in a low number of germ cells and inadequate germ cell maturation, simply repositioning the testis into the scrotum will not correct the endocrinopathy. Hence, supplementary treatment should be considered for boys with evident hypoplasia of germ cells and malfunctioning of the hypothalamic–pituitary–gonadal axis. Moreover, if the boy has a severely decreased number of germ cells, it may be valuable to offer testicular tissue cryopreservation of the early germ cells, while they are still present. One may advocate that germ cells are the most unique and precious cell type of the human body. They not only proliferate and differentiate but also are the only cell type to undergo meiosis to produce haploid gametes, having the ability to give rise to each subsequent generation. Testicular cells serve a crucial role in germ cell differentiation and maintenance including the production of reproductive hormones. The production of the haploid spermatozoa is a highly complex process, in preparation for embryonic life involving the migration of the bipotential primordial germ cells to the formation of fetal, neonatal, and prepubertal germ cells, which further develops from puberty and continues through the renewal and division of spermatogonial stem cells (SSCs) to produce spermatozoa by meiosis. The sex-specific development of the male germline initiates around 6–7 weeks post-conception with the expression of sex-determining region Y (SRY) and SRY-box transcription factor 9 (SOX9) in the gonadal somatic cells (pre-Sertoli cells), inducing the formation of seminiferous cords with Sertoli cells [23-25]. With their formation, Sertoli cells start to synthesize anti-Müllerian hormone (AMH), which inhibits Müllerian duct development and thereby prevents female development [24]. At this point, primordial germ cells are settled in the gonadal ridge and are now commonly referred to as gonocytes, where they begin to differentiate and proliferate populating the seminiferous cords along with Sertoli cells. In this thesis, the term gonocyte covers all germ cells after they become residents in the developing testis [24]. Other classifications of gonocytes and fetal germ cells, for example, pro- or prespermatogonia have been proposed [26, 27] but no ubiquitous consensus regarding terminology has been reached. Leydig cells begin to appear within the interstitial of the testis around week 9 post-conception [23, 24]. They secrete testosterone and other factors critical for germ cell differentiation and testicular development such as insulin-like-3 hormone (INSL3). Surrounding the seminiferous cords lie peritubular myoid cells, factor-secreting muscle cells, that can be recognized at week 12 post-conception [23, 24]. During the first trimester, gonocytes are mitotically active forming a quite homogenous cell population expressing markers typical of pluripotent cells including primordial germ cells such as KIT proto-oncogene (c-KIT), octamer-binding transcription factor 3/4 (Oct3/4, also known as POU5F1), lin-28 homolog A (LIN28), homeobox protein Nanog (NANOG), anti-podoplanin M2A antigen (D2-40, also referred to as M2A), and placental alkaline phosphatase (PLAP) [23, 24, 28-32]. This suggests that gonocytes are quite equivalent to primordial germ cells, both presenting the distinctive morphology of being large circular cells with a prominent nucleus containing one or two nucleoli surrounded by a spherical shape cytosol [26, 33]. Gonocytes occupy the center of the lumen-less seminiferous cords and are easy to distinguish from the associated Sertoli cells (Fig. 1B). Based on stereological estimations of 50 fetal human testes, the number of germ cells increased from a mean of 3.700 to 1.417.000 from 5 to 19 weeks post-conception [34]. Toward the end of pregnancy, the majority of fetal germ cells lose mitotic activity together with the pluripotency and fetal markers starting to form different germ cell subpopulations. They transform into fetal spermatogonia which are larger, flattened cells located on the basement membrane [1]. Simultaneously, they begin to express additional germ cell-specific markers such as melanoma-associated antigen A4 (MAGE-A4), dead-box helicase 4 (DDX4; also known as VASA), and deleted in azoospermia like (DAZL) [28, 30]. In support of this, Li et al. [30] studied testes from 12 human fetuses ranging from 4 to 25 weeks of gestation for single-cell RNA sequencing and grouped fetal germ cells into three clusters: "migrating," "mitotic," and those entering "mitotic arrest." The cluster of the "migrating" fetal germ cells was dominated by the expression of Oct3/4, NANOG, Sal-like 4 protein 4 (SALL4) whereas the "mitotic" fetal germ cells expressed the same but in addition VASA and DAZL. From week 9 and onwards, "mitotic arrest" fetal germ cells appeared to have similar expression patterns, but weaker for Oct3/4 and NANOG and stronger for VASA and DAZL in addition to new markers. Importantly, in the testis from an 18-week of gestation fetus, VASA-positive germ cells were distributed more in the peripheral zones of the seminiferous tubules in contrast to the Oct3/4-positive germ cells. Altogether, this supports the notion that male fetal germ cells develop through stages of migration, mitosis, and cell-cycle arrest. Postnatally, the germ cells continue to undergo further differentiation and migration toward the basement membrane of the seminiferous tubule. After migration, the morphology of germ cells is distinctly different forming the spermatogonial population, which consists of SSCs and other non-stem cells progenitors (subtypes of spermatogonia, Fig. 1B,C). Three morphologically distinct types of spermatogonia have been classified, namely type A dark (Ad), type A pale, and type B spermatogonia [35-37]. It is believed that gonocyte transformation into type A dark (Ad) spermatogonia is an essential step for the formation of the SSC pool necessity for fertility [38, 39]. In other words, Ad spermatogonia are considered to represent the SSC dividing to either self-renew to maintain the SSC pool or give rise to the Ap spermatogonia that may undergo one or more divisions before giving rise to differentiated B spermatogonia [36]. The nucleus of the Ad spermatogonia is homogeneous dense and dark with at least one rarefaction zone (chromatin-free cavity), which is distinguishable from the lightly stained, coarser nuclei from the other two types without the rarefaction zones (Fig. 1A) [36, 37, 40]. Ad spermatogonia usually appear during minipuberty, sharply increasing in number by the age of 4–5 months and remaining largely quiescent [41, 42], until the boy enters puberty where germ cell differentiation begins, truly indicated by a transition capable of meiotic division [24]. Studies have demonstrated that some fetal germ cells seem to remain after birth and along up to the first months of life, as demonstrated by some germ cells positive for the maintaining markers D2-40, Oct3/4, c-Kit, and NANOG [43-47]. Kvist et al. [44, 45] found positivity for D2-40 in germ cells up to 6 months of age, Oct3/4 up to age 6 and 9 months, and c-Kit up to 11–16 years during puberty. Gonocytes that fail to migrate to the basement membrane and differentiate normally undergo apoptosis and are cleared from the seminiferous epithelium, which presumably takes place during minipuberty until 1 year of age [37, 48, 49]. The importance of Ad spermatogonia has been supported by follow-up demonstrating their link with fertility outcomes [39, 50]. Ad spermatogonia are sometimes positive for PLAP and undifferentiated embryonic cell transcription factor 1 (UTF1) [51, 52], however, no specific marker for only Ad spermatogonia exists [53]. More recent publications including novel omics techniques have made attempts to assign spermatogonial subtypes into a hierarchical organization or functional stages [30, 46, 54, 55], indicating that SSC might have more complex physiology than previously assumed. But despite these efforts to widen the landscape of the testis, no specific marker and unequivocal definition of the prepubertal human SSC have been made. Therefore, the morphological criteria for identifying Ad spermatogonia have been used in the present thesis. Transformation of spermatogonia to primary spermatocytes usually begins around the age of 3–4 years [37, 38]. During childhood, the germ cells express various different markers, such as c-KIT, UTF1, PLAP, MAGE-A4, VASA, and fibroblast growth factor receptor 3 (FGFR3) but normal materials during the prepubertal period are sparse [44, 47, 55]. Germ cells together with Sertoli cells are most prominent in the prepubertal testis. Sertoli cells appear to have a more constant morphological and immature appearance from birth to 11 years of age, in contrast to Leydig cells that are more scarce and only seldom demonstrable in the light microscopy after 2 years of life [37, 42, 48]. The prepubertal testis may be regarded as a quiescent organ but serves as a crucial period for germ cell development. Given the highly complex environment necessary for germ cell development requiring delicate cellular arrangement, cell-to-cell interactions, migration, mitosis, and possible endocrine factors, the prepubertal period is highly sensitive and at risk of being disturbed, which consequently can lead to disease and infertility. Moreover, proliferation and differentiation take place in the prepubertal testis as the number of germ cells varies a lot from mid-gestation toward puberty [56]. During the period from week 28 of gestation until around 3 years of life, a rise in the total number of germ cells by a factor of 3 was demonstrated during the first 100 days of life with a maximum at 100–150 days of age and followed by a decreased by a factor of 0.5 [49]. From the same materials, Sertoli cells increased by more than a factor of five during the first 3 months of life to reach a steady density until the onset of puberty where Sertoli cells increased by a factor of 2 [57]. The postnatal escalation of Sertoli cells elongates the seminiferous tubules and results in testicular growth [58, 59]. The histological parameter "number of germ cells per cross-sectional tubule (G/T, also referred to as S/T; 'S" stands for spermatogonia) has been used in the quantification of germ cells and correlates with the number of germ cells per cm3 testicular parenchyma [56]. Hence, G/T is a parameter of numerical density. Based on normal materials, it has been demonstrated that G/T decreases slightly from birth to 3 years of age, with the most rapid decline during the first year, and then increases afterward until 8 or 9 years with a small decline or pause to then increase markedly toward the puberty [37, 56, 60, 61]. The prepubertal testis has two main functions—the production of sex steroids and the establishment and maintenance of the diploid germ cells, essential for male genital differentiation, growth, and future spermatogenesis. At the onset of puberty, the organization of the prepubertal testis changes dramatically as the lumen occurs and Sertoli cells mature and form the blood–testis barrier by compartmentalizing the seminiferous epithelium into basal and ad luminal compartments [1]. These changes enable spermatogenesis. The two pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the pivotal endocrine regulators of these testicular functions controlled by pulsatile secretions of gonadotropin-releasing hormone (GnRH, also known as the luteinizing-releasing hormone, LHRH) from the hypothalamus. This system is coordinating a tightly regulated feedback loop between the hypothalamus, the anterior pituitary, and the testes, the so-called hypothalamic–pituitary–gonadal axis. Evidence suggests that early stimulation of the testis is regulated by the placental human chorionic gonadotropin (hCG) since hypothalamic control of gonadotropic function is not operative until after the first trimester [62, 63]. The hypothalamic–pituitary–gonadal axis is largely quiescent until the onset of puberty, except for two brief transient activations; one during fetal life at mid-gestation and a second activation at 2–5 months postnatally known as the minipuberty [64, 65]. During minipuberty, circulating testicular hormones reach high levels as seen in adults [66]. The gonadotropins exert differential targets on the compartments of the testes. LH stimulates Leydig cells that secrete testosterone, along with glycoprotein mediators such as INSL3, whereas FSH presumably targets Sertoli cells to release inhibin B and anti-Mullerian hormone (AMH) [67]. A Danish study including reproductive hormone measurements on 1840 infants (1041 boys and 799 girls) during minipuberty showed that the LH concentrations overcome the FSH concentrations in male infants, the opposite situation occurs in females [68]. It is generally believed that minipuberty is essential to prime germinal epithelium for subsequent germ cell maturation as well as expansion since the transient peak is associated with increased proliferation of Sertoli cells, Leydig cells, and germ cells [42, 48, 49, 57, 69]. Of importance, it is believed that minipuberty is essential for the gonocyte transformation into Ad spermatogonia [48, 70]. Moreover, the postnatal surge of testosterone is believed to be associated with a rapid increase in penile growth, testicular volume, and anogenital distance [65, 71, 72]. Despite the presence of high testosterone and FSH, spermatogenesis does not progress since immature Sertoli cells are devoid of androgen receptors. Minipuberty may also have an important impact on secondary sex characteristics and other biological functions [65, 73]. After 6 months of age and through childhood, LH and testosterone decrease to very low or undetectable concentrations [66] (Fig. 2). FSH and inhibin B also decrease but remain clearly detectable throughout childhood [66] (Fig. 2). In contrast, AMH remains rather high during childhood [74] until it decreases in puberty [75]. The normal process of testicular descent is multi-staged and complex involving the interplay of anatomical and hormonal factors [76]. The current theory generally focuses on hormonal aspects and divides testicular descent into two functional phases: the "