Neuroendocrine Control of Reproductive Function PDF

**NEUROENDOCRINE CONTROL OF REPRODUCTIVE FUNCTION** The **hypothalamus** contains neurons whose axons end near the **anterior pituitary gland**, specifically in the **median eminence or pituitary stalk**. These neurons release hormones into a network of blood vessels (**portal system**), which tran...

**NEUROENDOCRINE CONTROL OF REPRODUCTIVE FUNCTION** The **hypothalamus** contains neurons whose axons end near the **anterior pituitary gland**, specifically in the **median eminence or pituitary stalk**. These neurons release hormones into a network of blood vessels (**portal system**), which transport them to the anterior pituitary. When reproductive hormones are needed, the hypothalamus releases gonadotropin-releasing hormone (**GnRH**). GnRH is released into the portal system and carried to the anterior pituitary. In the anterior pituitary, GnRH binds to its receptor (a G-protein-coupled R) in the gonadotrope cells. This triggers a signaling cascade through Gαs proteins, leading to an increase in cAMP, which activates PKA. This ultimately results in the production of two key hormones: follicle-stimulating hormone (**FSH**) and luteinizing hormone (**LH**), which are collectively known as **gonadotropins**. → Higher GnRH pulsatility → LH beta expression All steroid hormones, including estradiol and progesterone, are derived from cholesterol, which can be converted into several different molecules such as testosterone, estradiol, and progesterone. Both **ES** and **PG** provide **feedback** to the **hypothalamus** to regulate gonadotropin production, with stronger feedback effects on the hypothalamus and lesser effects at the level of the pituitary. Depending on the phase of the cycle, these hormones can have either inhibitory or stimulatory effects. Interestingly, GnRH neurons do not express receptors for estradiol, testosterone, or progesterone. So how do these hormones control GnRH activity? They exert their effects on an upstream regulatory system called the \"**kisspeptin system**.\" Kisspeptin neurons, which do express receptors for these steroids, in turn regulate GnRH neurons. The control of GnRH through the kisspeptin system is highly **dynamic** and changes based on the **levels** and **duration** of **exposure** to hormones like estradiol and progesterone. This system is flexible, allowing for precise regulation of reproductive function. There are other proteins produced by the mature follicle and the testes that influence activity at the pituitary level, either by stimulating or inhibiting it. **Activin** stimulates pituitary function, while **follistatin** prevents activin from exerting its effects, thus inhibiting its activity. **Inhibin** is also a key protein produced by growing follicles, and it works alongside [estradiol] to suppress FSH once follicle growth has been stimulated by the initial rise in FSH. Hormonal actions can be classified as **endocrine** (acting on distant cells), **paracrine** (acting on nearby cells), or **autocrine** (acting on the same cell that produced the signal). Estradiol plays a key role in facilitating the production of progesterone by activating enzymes that sequentially metabolize cholesterol into progesterone. **Theca cells** primarily produce **androgens** (such as testosterone) when stimulated by LH. The enzyme pattern in theca cells ensures that **testosterone** is consistently produced. The process begins with cholesterol, which is imported into the cell and cleaved in the mitochondria. Various enzymes then convert cholesterol into androgens. Theca cells generate a significant amount of androgens, a small portion of which can enter circulation, while most are transported to granulosa cells, where they are converted into estradiol. In **granulosa cells**, **estradiol** is produced. However, testosterone produced by the theca cells can be converted into estradiol by the enzyme aromatase, which is highly active in granulosa cells. **Androgens→ estradiol** The hypothalamus, through the kisspeptin neuron system, is highly sensitive to various factors that can influence the activity and patterns of these neurons, which are critical for regulating reproductive hormones. For example, low levels of leptin, as seen in women athletes with very low body fat, can lead to amenorrhea (the absence of menstruation). Adequate levels of body fat are necessary for normal reproductive function because adipocytes (fat cells) produce leptin when the body\'s fat and metabolic balance is stable. When metabolic demand is high and body fat is low, adipocytes reduce leptin production. Leptin signals to the kisspeptin neurons, which in turn help regulate the release of FSH (follicle-stimulating hormone) and LH (luteinizing hormone) from the pituitary gland. This mechanism is an evolutionary adaptation, ensuring that reproduction is suppressed during times of energy deficiency. Exogenous opioids (like pain medications) and cortisol (produced during stress) can also interfere with hypothalamic function. These substances can block the hypothalamus, disrupting its control over the reproductive system and reducing the release of GnRH (gonadotropin-releasing hormone), which is necessary for FSH and LH production. In addition to its role in reproduction, the hypothalamus regulates anxiety and hunger. It contains glucose receptors that detect blood sugar levels, and these signals can also influence the reproductive system. For instance, during times of stress or hunger, reproductive processes may be downregulated to conserve energy or due to the effects of stress hormones, further showing how closely linked metabolism, stress, and reproduction are. **Follicle selection** Angela Baerwald. Canada Follicles grow in response to rising FSH levels in the bloodstream. About five days after menstruation, FSH levels increase, stimulating follicular growth. This marks the beginning of the follicular wave. FSH is the hormone responsible for supporting follicle growth up to the point of **deviation**, after which LH takes over to support the growth of the dominant follicle. Without LH, the dominant follicle cannot continue growing, and ovulation will not occur. Follicles typically **grow until about 8 mm in size**, and without sufficient LH, they will regress (LH dependent phase). Similarly, without FSH, follicles cannot grow beyond 5 mm (FSH dependent phase).  Follicular waves in the female ovary involve a group of follicles (**cohort**) that grow together, but only one follicle will eventually ovulate during each cycle. This process is **follicle selection**. Although the process of follicle selection begins early, it isn't visible until the dominant follicle starts to outgrow the others. So the **follicle deviation** is the point where we are able to [observe] that one follicle becomes the dominant one, while the others (**subordinate follicles**) stop developing and regress (atresia). Deviation occurs when the follicle reaches about 7 mm in size **T2- GONADAL DEVELOPMENT AND EARLY GAMETOGENESIS** **How Follicles Are Formed and Activated** The **migration of primordial germ cells** leads to the formation of **testes and ovaries**. During this migration, germ cells differentiate under the influence of various factors that guide and regulate their development as they move. Among the most important factors are **BMP4** (Bone Morphogenetic Protein 4) and **BMP2** (Bone Morphogenetic Protein 2). Interestingly, the same growth factor that regulates bone development also governs these specific processes in the gonads. Throughout this stage of embryonic development, different cells within the embryo begin to differentiate and form primordial germ cells, in interaction with the endoderm. BMP4 and BMP2, play a crucial role in the differentiation of these cells and also play a key role in the differentiation of somatic or pluripotent cells into gametes. - - **Methylation and Demethylation** Pluripotent cells need the ability to differentiate into all necessary cell types. To achieve this, these cells must first acquire the genetic capacity to differentiate into any type of cell. **Demethylation opens** up the possibility for gene expression, allowing primordial germ cells to differentiate into various cell types. As these cells differentiate locally, they will gradually acquire the expression of genes specific to the cell type they are becoming. On the other hand, **methylation inhibits gene promoters**, preventing transcription. This regulates which genes are expressed and which remain silenced, thereby controlling the process of cellular differentiation. At the **histone level,** there are several mechanisms that regulate gene expression: - - - During follicle formation, oogonia (immature egg cells) are initially surrounded by undifferentiated granulosa cells. These early granulosa cells provide support to the oogonia. A large number of oogonia are produced through rapid proliferation, but many also undergo regression or degeneration before they are activated, making the mechanisms controlling these phases crucial for ovarian development. The surviving oogonia become surrounded by flat, immature granulosa cells, forming primordial follicles, which represent the earliest stage of follicle development. At this point, meiosis begins in the oogonia, though it pauses until further activation later in life. **Oogenesis** Oogenesis process begins with the ovarian stem cells, or **oogonia**. Oogonia are formed during fetal development and divide via mitosis, much like spermatogonia in the testis. Unlike spermatogonia, however, **oogonia forms primary oocytes in the fetal ovary prior to birth**. These primary oocytes are then **arrested in this stage of meiosis I**, only to resume it years later, beginning at puberty and continuing until the woman is near menopause (the cessation of a woman's reproductive functions). The number of primary oocytes present in the ovaries declines from one to two million in an infant, to approximately 400,000 at puberty, to zero by the end of menopause. The initiation of ovulation---the release of an oocyte from the ovary---marks the transition from puberty into reproductive maturity in females. From then on, throughout the reproductive years, ovulation occurs approximately once every 28 days. Just prior to ovulation, a surge of LH triggers the resumption of meiosis in a primary oocyte. This initiates the transition from primary to secondary oocyte. However, as shown in the figure below, this cell division does not result in two identical cells. Instead, the cytoplasm is divided unequally, and one daughter cell is much larger than the other. This larger cell, the secondary oocyte, eventually leaves the ovary during ovulation. The smaller cell, called the first polar body, may or may not complete meiosis and produce second polar bodies; in either case, it eventually disintegrates. Therefore, although oogenesis produces up to four cells, only one survives.  **Meiosis** - - In the development of oocytes, meiosis results in one functional egg cell and two polar bodies that are extruded. This process involves the exclusion of these polar bodies, starting with the loss of one of the homologous chromosomes. The second chromosome is only excluded if fertilization occurs after a peak in luteinizing hormone (LH), which triggers meiosis II. If the LH peak does not occur, the oocyte remains in prophase I of meiosis II. In assisted reproduction, the focus is on obtaining metaphase II (MII) oocytes, which can be identified by the presence of the first polar body, indicating that they have completed meiosis I in response to LH stimulation. If fertilization takes place, the second polar body, which contains one of the chromatids, is also excluded, ensuring that the resulting embryo has the correct chromosome number. **Aneuploidy** is a genetic condition that occurs due to errors during meiosis. Aneuploidy can arise in either Meiosis I, where homologous chromosomes fail to separate properly, or in Meiosis II, where sister chromatids do not separate, both leading to conditions that involve an abnormal number of chromosomes in the resulting cells. If errors occur during the MI separation, it can lead to aneuploidy, where gametes end up with an abnormal number of chromosomes. For instance, trisomy of chromosome 21 leading to Down syndrome.  **The spindle assembly checkpoint (SAC)** is a crucial regulatory mechanism that helps minimize errors during cell division, particularly during meiosis. The SAC is a collection of proteins that monitor the attachment and tension of microtubules on chromosomes and sister chromatids. The centrosome plays a key role in this process, as it is where microtubules anchor to pull chromosomes apart during cell division. The SAC senses whether the traction on these chromosomes is balanced. If the tension is not appropriately balanced, the SAC will prevent the progression into anaphase, the stage where chromosomes are separated and pulled toward opposite poles of the cell. The SAC remains active until the necessary tension is achieved, ensuring accurate chromosome segregation. Once this balance is confirmed, the SAC is inhibited, allowing anaphase to proceed. This process requires energy and is sensitive to oxidative stress; if oxidative stress levels are high, the SAC may malfunction. Additionally, mitochondria produce reactive oxygen species (ROS), and if these accumulate excessively, they can disrupt the SAC\'s function. Furthermore, elevated levels of follicle-stimulating hormone (FSH) can also interfere with this checkpoint mechanism, potentially leading to errors in chromosome segregation. As women age, particularly in advanced maternal age, there is an increase in FSH levels. FSH is a hormone crucial for regulating the growth and maturation of ovarian follicles, which contain the oocytes (egg cells). The elevated FSH levels associated with aging can disrupt the communication between the oocyte and the surrounding cumulus cells. This breakdown in communication can lead to a decline in oocyte quality, affecting fertility and increasing the risk of chromosomal abnormalities in eggs. Interestingly, studies have shown that the levels of aneuploidy (the presence of an abnormal number of chromosomes) in oocytes correlate with FSH levels throughout a woman's life. FSH levels tend to be higher during prepuberty and postpuberty and rise again in later reproductive years. This pattern suggests that as FSH increases, especially in the context of advanced maternal age, the likelihood of aneuploidy also increases, further contributing to fertility challenges and potential genetic issues in offspring. **What induces follicle activation?** Follicles are gradually activated throughout a woman\'s life, but not all follicles are activated simultaneously. The activation of specific follicles at certain times is influenced by various factors that either promote or inhibit this process. One of the key players in follicle activation is granulosa cells, which help \"wake up\" oocytes (egg cells). These cells are sensitive to their local environment, particularly to levels of energy, oxygen, and metabolic conditions. When conditions are favorable---such as increased oxygen levels and glucose metabolism---the activity of the mTOR signaling pathway increases. mTOR functions as a transcription factor that regulates cell growth and metabolism. When **mTOR** is activated in a particular region of the ovary, it **signals granulosa cells to become active**, which in turn helps activate the oocytes. Once activated, granulosa cells produce paracrine factors that create a signaling loop. One important factor is **kit ligand (Kitl)**, a protein secreted by granulosa cells that activates receptors on the oocyte called Kit receptors. When these receptors are activated, they trigger a cascade of signaling pathways, notably the **PI3K** (phosphoinositide 3-kinase) signaling pathway, which is crucial for oocyte growth and maturation. However, there are also **inhibitors** present in this process. Proteins such as **FOXO3** and **PTEN** act as negative regulators of the PI3K pathway, which can inhibit follicle activation and oocyte maturation. This balance between activation and inhibition is essential for ensuring that only the appropriate follicles are activated at the right times during a woman\'s reproductive life. -  Once the oocyte is activated, it begins to stimulate the granulosa cells through a bidirectional communication process. This interaction is crucial for the development of both cell types. Activated oocytes produce two important factors: Growth Differentiation Factor 9 (**GDF9**) and Bone Morphogenetic Protein 15 (**BMP15**). Both of these factors are essential for the **continued maturation** and **development** of **granulosa cells**, which are vital for supporting the oocyte. If there is a **knockout** of either GDF9 or BMP15, it results in a complete **failure of follicle development** from the early stages. Specifically, primary follicles will not progress or grow further, highlighting the critical role these factors play in the growth and maturation of ovarian follicles. Thus, the interaction between the oocyte and granulosa cells, mediated by GDF9 and BMP15, is essential for successful follicle development. - - **FGF-2 and KL stimulate follicle activation** Paracrine growth factors play a crucial role in stimulating follicular growth and activation. Fibroblast Growth Factor 2 (**FGF2**) is expressed by the oocyte of primordial and primary follicle and is known to stimulate both **follicle activation** and **growth**. FGF2R is in **granullosa cells.** In an experiment designed to investigate these effects, four conditions were tested: a control with fresh ovarian tissue, a control with treatment lacking growth factors, and two treatments---one with kit ligand (Kitl) and the other with FGF2. When ovarian fragments were treated with kit ligand, there was a noticeable decrease in the number of primordial follicles, accompanied by an increase in the number of developing follicles. A similar effect was observed with FGF2 treatment. Both factors effectively stimulate follicle activation, leading to a reduction in the resting pool of primordial follicles and an increase in the developing pool of follicles. **FGF7 stimulate follicle activation and has positive feedback with KITL** **FGF7 or KGF** is necessary for the full function of KITL, as its suppression leads to a loss of the stimulating effects of Kitl. Kitl enhances the expression of FGF7, creating a **synergistic** relationship between the two factors, whereby both stimulate each other\'s expression and further promote follicular activation.  **Androgens play a role in stimulating the transition of primary follicles into secondary follicles** Androgens can be produced in the ovaries by inducing theca cells. Secondary follicles are characterized by having multiple layers of granulosa cells. Research has shown that testosterone and other androgens can enhance the expression of FSH receptors in small follicles, which makes them more responsive to subsequent FSH treatment. **AMH inhibits follicle activation** Anti-Müllerian hormone (AMH) serves as a crucial **marker of ovarian reserve**. Higher levels of AMH correlate with an increased number of growing follicles, as it is produced by the granulosa cells of activated and growing follicles. Oogonia undergo a phase of rapid proliferation, during which they also experience significant degeneration. The size of the ovarian reserve is closely linked to this balance. The mechanisms of proliferation and degeneration are the same for all women, but they differ in intensity. Women who experience greater proliferation and less degeneration will have a larger ovarian reserve, leading to a later onset of menopause. AMH also functions as a vital paracrine **regulator of folliculogenesis.** Its primary role is inhibitory, aiming to maintain a balance in follicle activation. Ovarian follicles cannot all be activated simultaneously; rather, this process must occur gradually. **AMH inhibits the activation of primordial follicles** and plays a role in modulating the **transition from primary to early antral follicle** stages. AMH downregulates key signaling pathways involved in follicle development. It can **inhibit Kitl, GDF9, BMP15, and FGF** signaling, all of which are critical for follicular growth and activation. By regulating these pathways, AMH ensures that follicular development occurs in a controlled and balanced manner, preventing premature activation of all follicles in the ovary. During the antral phase of follicular development, AMH serves as a **protective factor against excessive levels of FSH**. This action helps to prevent overstimulation of the follicles, ensuring a balanced environment for healthy follicle development. Interestingly, the oocytes that are most likely to lead to successful pregnancies are those with higher levels of AMH and lower levels of FSH. Elevated **AMH indicates robust follicular health**. **FOLLICULOGENESIS AND THE OVULATORY CASCADE** Follicle development occurs in waves, rather than as a random process. During the preantral phase, follicular growth is largely independent of gonadotropins (hormones like FSH and LH). Instead, local peptides regulate follicle activation and the transition to the antral stage. Once follicles reach the antral stage, however, gonadotropins become essential for further development. A key stimulus for follicle growth is the rise in follicle-stimulating hormone (FSH) that occurs before the onset of the follicular wave. This increase in FSH is what triggers follicle recruitment, which refers to the process by which a group of follicles (cohort) is selected for growth. Under the influence of FSH, these follicles develop for about two days, growing until they reach a size of 8-10 mm. At this point, only one follicle continues to mature and proceed toward ovulation. The process of \"deviation\" marks the visible stage where one follicle is selected as the dominant follicle, while the others undergo **atresia** (degeneration). This selection leads to two distinct phases: **FSH dependence and LH dependence.** The dominant follicle is able to survive even in low FSH conditions, whereas the non-dominant follicles regress without sufficient FSH. **IS LH RESPONSIBLE FOR FOLLICLE DEVIATION?** - In the image, we see granulosa and theca cells, and we are specifically focusing on the **LHR**, which is initially only present in theca cells. LH stimulates these theca cells to produce androgens. Once the theca cells develop, they begin to express LH receptors. In the early, very small follicles that have just been recruited, LH receptors are only found in theca cells, not in granulosa cells. However, in **selected oocytes** (those moving toward dominance), there is a **small amount of LH receptor** expression in granulosa cells. Some experts believe that the ability of these follicles to respond to LH may be a key factor in determining which follicles will become dominant. Therefore, the follicles that begin expressing LH receptors in their granulosa cells might be the ones that go on to become dominant. However, there are **6 different isoforms** of the LH receptor. The fully **functional isoform (Isoform 1)** is responsible for the complete function when it is translated into a protein. The other 5 isoforms are truncated, meaning they have deletions in certain regions and may not be fully functional. For instance, if a male has a truncated isoform of the LH receptor, he may lack male characteristics due to insufficient testosterone production. In bovine models, it has been observed that in theca cells, Isoform 1 (black) is always expressed. In contrast, in **granulosa** cells, this isoform **appears** only around the time of deviation, but in very small amounts, and becomes more prominent **after deviation**. Deviation occurs when the follicle reaches about 7 mm in size. The appearance of the LH receptor in granulosa cells after deviation likely plays a role in allowing the follicle to progress toward ovulation, rather than being involved in the selection process itself. So, is LH receptor expression in granulosa cells important during follicle selection? Possibly not, as the presence of mRNA for the LH receptor doesn\'t necessarily mean the receptor is functional. Therefore, it might not be a crucial factor in the initial selection of the dominant follicle. GC express LH receptors after deviation. Under LH stimulation, they express progesterone which is essential for ovulation. **WHAT MAKES THE DOMINANT FOLLICLE RESISTANT TO THE ABSCENCE OF FSH?** One major factor is the activity of the insulin-like growth factor **(IGF) system**. IGFs, especially IGF-1, stimulate the production of **estradio**l (ES) and the **proliferation of granulosa cells.** If IGF-1 is removed from the follicles, the follicles fail to survive. There are two key ligands in this system: **IGF-1**, which is produced in the liver and reaches the follicle through circulation, and **IGF-2**, which is produced locally by theca cells. Both IGF-1 and IGF-2 act on **receptors** that are present in both **granulosa** and **theca** cells. However, the **availability** of IGFs is tightly regulated by a group of proteins known as IGF-binding proteins (**IGFBPs**). These proteins are crucial for **transporting** IGFs but also for controlling how much IGF is available in the follicle's local environment. When IGF is bound to IGFBP, it is **inactive** because it cannot bind to its receptor. **In dominant follicles, there is less IGFBP present.** The amount of IGFBP in the follicle is regulated by an enzyme called **PAPP-A**, which **cleaves IGFBP.** **The dominant follicle has higher levels of PAPP-A**. As the follicle continues to develop, LH receptors eventually appear, allowing LH to support the follicle's growth and guide it towards ovulation, completing its final maturation. At this stage, **meiosis resumes** within the oocyte. We must also consider **inhibitory factors** in follicle development. While the dominant follicle benefits from the IGF system, which promotes growth by providing high levels of stimulatory factors and fewer inhibitors, subordinate follicles have the opposite balance---fewer growth-stimulating factors and more inhibitory ones. One key inhibitory factor is fibroblast growth factor 10 (**FGF10**), which inhibits follicle growth by **reducing estradiol** (ES) production. Since estradiol is essential for follicle development, any factor that reduces its production is considered inhibitory. In lab experiments, granulosa cells normally produce estradiol when cultured. However, when granulosa cells are cultured with FGF10, they stop producing estradiol. This demonstrates the inhibitory role of FGF10. To test this further, researchers injected FGF10 into follicles in vivo (inside the body) during the phase when an oocyte was selecting the dominant follicle. They used both small and high concentrations of FGF10, along with PBS as a control to ensure that the effects were not due to the physical damage of the injection. The results showed that even a small dose of FGF10 caused the dominant follicle to stop growing and become subordinate. At higher doses, estradiol production dropped dramatically, further inhibiting follicle growth. In the graph, the dominant follicle is shown in black, with the subordinate follicle alongside it. The **deviation** stage in cows occurs **around day 2.5.** Just before deviation, on day 2, the dominant follicle shows a decrease in the expression of FGF10. In the other graph, we see the **receptors for FGF10.** At the time of **deviation**, the subordinate follicle **expresses more FGF10 receptors** than the dominant follicle. **This means the subordinate follicle not only produces more FGF10 but is also more sensitive to it, due to the higher expression of its receptors.** **OOCYTE MATURATION AND MEIOSIS RESUME** The major factor driving the **final development** of the follicle for ovulation is **LH** The oocyte itself cannot complete maturation until the precise time during follicle development. Meiosis is paused in prophase I of **meiosis** I, and it will only **resume after the LH peak.** This process only occurs in the dominant follicle because the subordinate follicles do not respond to the LH peak and never reach this stage of development. **So why doesn\'t the oocyte mature until the LH surge, and how does LH restart meiosis?** In the lab, we aim to obtain oocytes at the MII(metaphase II) stage, which indicates full maturation. However, achieving MII is not sufficient by itself. Completing meiosis is relatively simple; the real challenge lies in **the timing of the oocyte's cytoplasmic preparation** while it is being held in the arrested stage of meiosis. The oocyte cannot mature prematurely because its cytoplasm is not yet fully prepared. During the **oocyte's growth** phase, even though the nucleus is not maturing, the oocyte is actively **preparing the cyto**plasm. Once the oocyte enters maturation, the chromatin condenses, which **halts transcription activity.** No more proteins can be produced at this stage. **Transcription is crucial for maturation**, so if we want the oocyte to accumulate the necessary proteins for development, we must avoid premature chromatin condensation. The key is to allow the oocyte time to prepare and mature naturally---rushing this process can be detrimental. Now, turning to a key experiment published in Science: granulosa cells play a critical role in this process. This experiment explains how the oocyte is arrested in prophase I. In the first image A, we see an oocyte in the germinal vesicle (GV) stage, which is loose and should remain in this stage until the LH peak. At this point, the follicle is growing, and the oocyte is still maturing. The chromatin is surrounded by an envelope and remains uncondensed, allowing transcription to continue. In the next image B, we see the results of **knocking out the NPPC receptor and NPPC ligand.** NPPC (natriuretic peptide precursor C) is an important peptide produced in various tissues, including the follicle. With the NPPC knockout, the oocyte prematurely reaches the MII stage. The germinal vesicle envelope degenerates, and the genetic material condenses. This is abnormal, as the **oocyte should remain in the GV stage until the LH peak**. NPPC is a key factor in maintaining the oocyte\'s arrest in prophase I, preventing premature maturation. **HOW THE OOCYTE IS ARRESTED IN PROPHASE I** We start with the oocyte in the germinal vesicle (GV) stage, where a key factor in maintaining the arrest in prophase I is **MPF** (Maturation Promotion Factor), a complex that, when activated, induces maturation. However, MPF is **continuously inhibited by cAMP** (cyclic adenosine monophosphate). High levels of cAMP are necessary to hold the oocyte in prophase I by inhibiting MPF activation. In the diagram below, there is a receptor that is constitutively active (meaning it remains active without a ligand) and produces cAMP (red) continuously. However, there is a challenge: **PDE3A** (phosphodiesterase 3A) is always present and works to degrade cAMP. To maintain the prophase I arrest, we need to prevent PDE3A from degrading cAMP. PDE3A has a higher affinity for **cGMP** (yellow) than cAMP, so cGMP essentially distracts PDE3A, preventing it from degrading cAMP. We can\'t completely knock out PDE3A because doing so would permanently block the oocyte in its arrested state, preventing any maturation. **cAMP SOURCE:** **NPPC activates** receptors in **GC and CC** and induces the **production of cGMP** in these cells creates a flow of cGMP from the **mural** granulosa cells through the **cumulus** cells, a special type of granulosa cells that are more closely associated with the oocyte and influenced by oocyte-secreted factors. This flow is regulated by **connexin 43**, a protein that facilitates communication between cells through gap junctions. The zona pellucida, a protective layer surrounding the oocyte, poses a barrier, but **transzonal processes**---projections from cumulus cells that extend through the zona pellucida---allow cGMP to travel from the cumulus cells to the oocyte. The **transzonal processes**, which extend from cumulus cells to the oocyte, not only deliver **cGMP** but also transport other essential materials, such as **RNA** ready for translation and **antioxidant** molecules like APH (anti-peroxide hydrogen). These materials are fundamental for ensuring the **oocyte's quality**. Once nuclear maturation is triggered, these transzonal processes close, cutting off the flow of supportive materials. Therefore, if maturation is triggered too early, it can harm the oocyte\'s development. If we stimulate the oocyte **prematurely**, we compromise its quality. **HOW LH TRIGGERS OVULATION AND MEIOTIC RESUMPTION** LH plays a crucial role in follicular development, specifically acting through the **mural granulosa cells which express LH receptors.** However, the **cumulus cells do not express LH receptors**. This means there must be mediators that carry signals from the mural granulosa cells to the cumulus cells and eventually to the oocyte. Under **LH stimulation,** the **mural granulosa** cells produce **AREG** (Amphiregulin) and **EREG** (Epiregulin). These factors **activate** receptors in the **cumulus cells.** These cumulus cells produce AREG and EREG and activate the ERK/MAPK pathway producing ERK1 and ERK2. **AREG and EREG** in the cumulus cells induce the **closure of the zonal processes** by **phosphorylation**. As a result, cGMP can no longer flow to the oocyte, and the production of cGMP also decreases.  In the next step, **ERK1 and ERK2** cause the **retraction of the transzonal projections**. This retraction results in the disconnection of the gap junctions, halting the flow of cGMP. With the **reduction of cAMP** levels, **MPF** is released and **activated**, allowing the **oocyte to resume meiosis**. ERK1/2 enhances the expression of different genes: - - - **THE OOCYTE DOESN\'T LIKE BEING PUSHED** - The oocyte exhibits a resistance to being pushed out prematurely during its development. In the context of this discussion, we will focus on the signaling molecules AREG and EREG. In our experimental setup, we have graphs depicting the levels of AREG and EREG. There is an image that we cannot see currently, but it will be provided later. This setup allows us to examine the bidirectional communication between the oocyte and cumulus cells. Specifically, we aspirate the oocyte, leaving only the cumulus cells. - - - We observe that the presence of the oocyte (Grey) decreases the expression of AREG and EREG; **the oocyte downregulates both AREG and EREG** by **secreting factors** that inhibit their production. Additionally, **FGF10** is also produced by the oocyte. In another graph, we examine the same three experimental groups, but this time with **NPPC**. The **oocyte appears to favor NPPC** through its secreted factors, suggesting that it is not in a hurry to complete meiosis. **Does the oocyte respond positively to FSH?** Cumulus cells do express FSH, but our findings indicate that the oocyte does not favor its presence. This is particularly curious, as FSH levels are generally increased during ovarian stimulation protocols. *J.E fortune. search that woman: this woman discovered that androgen signaling in preantral folicles actvate folicle transition.* **Progesterone Signaling and Follicle Ovulation** **Progesterone** signaling, along with **oxytocin** and **prostaglandin** signaling, is fundamental for **follicle ovulation**. These signaling pathways involve **metalloproteinases**, which are enzymes that **degrade the follicular wall**, causing it to rupture. This process is characterized by an inflammatory response followed by the degradation of the follicular wall. During the growth of the dominant follicle, granulosa cells initially do not express LH receptors. Instead, they produce estrogen (ES). However, after the selection of the dominant follicle and as it approaches ovulation, granulosa cells begin to express LH receptors. Under the influence of LH, these cells start to produce **progesterone**, which is essential for the ovulation process. The increased production of PG indicates that these cells are differentiating into luteal cells, as the corpus luteum is responsible for producing progesterone. Progesterone plays a complex role in the hormonal feedback loop. **Initially, they act as negative regulators during the luteal phase;** they exert a negative feedback effect at the level of the hypothalamus by inhibiting the kisspeptin system, which in turn decreases the release of GnRH from the hypothalamus and, consequently, **reduces FSH and LH** production from the pituitary gland. The hypothalamus is dynamic in its response to steroid hormones. When exposed to **chronic levels** of PG, the hypothalamus downregulates its **PG** receptors. Once **luteolysis** occurs, progesterone **levels drop**, and during the entire follicular phase the hypothalamus is not exposed to progesterone for a prolonged period (about 10 days). This lack of exposure to progesterone creates a different environment around the time of ovulation. Interestingly, just **after ovulation**, the follicles begin to differentiate into a corpus luteum. **Granulosa cells** within the follicle, upon exposure to rising **LH levels**, transition to a **luteal phenotype** and start **producing PG.** This early production of progesterone can have a positive effect, contrary to its traditional inhibitory role. **When the dominant follicle begins producing PG** for the first time, this triggers a **positive feedback** loop: kisspeptin neurons respond favorably to PG, promoting the release of GnRH. This mechanism is critical for **inducing the LH peak**, which is essential for ovulation. **Recent research has shown that progesterone, when administered at the right time, can induce ovulation by increase the LH surge.** **CORPUS LUTEUM** When the follicle ruptures, the oocyte is released into the fallopian tubes, but the follicle itself also plays a crucial role. **After ovulation**, it must differentiate into a **corpus luteum**, which produces **progesterone**. This hormone is essential for **supporting early embryonic development** and **preparing** the uterine environment **for implantation** and growth. When a follicle ovulates, it transforms into a **corpus luteum**, which alters the hormonal activity of the pituitary gland. Instead of producing estradiol, the corpus luteum begins producing **progesterone**. After ovulation, granulosa cells (GCs) transform into large luteal cells, while theca cells (TCs) differentiate into small luteal cells under the influence of LH. This transformation is driven by changes in enzyme expression; instead of producing estrogen, these cells begin producing progesterone. The corpus luteum has a very high metabolic rate and requires significant blood supply to function effectively. This increased demand leads to enhanced angiogenesis. Key angiogenic factors, such as **IGF**, **FGF**, and **VEGF**, play critical roles in this process. During the formation of the corpus luteum, blood vessels that previously supplied the theca layer expand and migrate into the developing corpus luteum, providing the necessary nutrients for progesterone production. The presence of **FGF2** and **VEGF** is particularly important for promoting blood vessel formation. However, if FGF2 is inhibited using an FGFR inhibitor, vascularization is significantly reduced, highlighting FGF2\'s critical role in angiogenesis. Some studies propose that **FGF2 is even more crucial** than VEGF, as VEGF alone cannot achieve sufficient angiogenesis without the presence of FGF2. One important enzyme involved in progesterone production is **STAR** (steroidogenic acute regulatory protein), which facilitates the transport of cholesterol into the mitochondria, a key step in steroidogenesis. Following ovulation, steroidogenesis shifts toward progesterone production, which is vital for maintaining the early stages of pregnancy. **MATERNAL RECOGNITION OR LUTEOLISIS** If fertilization does not occur, the corpus luteum undergoes **regression**, a process known as **luteolysis**. In the presence of an embryo, the embryo signals the corpus luteum to maintain its function, protecting it from regression with **human chorionic gonadotropin (HCG)**, which preserves the corpus luteum's production of **progesterone**. During a **fresh embryo transfer, ovulation is triggered using HCG** instead of LH. HCG has a prolonged effect in the human body, effectively rescuing the corpus luteum from regression. Specifically, HCG stimulates the production of PGE2, which in turn activates LH receptors while suppressing FSH receptors. Both **HCG and PGE2 work synergistically to enhance progesterone production** by stimulating the expression of essential enzymes. If there is no embryo, luteolysis occurs and menstruation is triggered by a drop in progesterone levels. Key factors involved in luteolysis include **BMP2, BMP4, and BMP6**, which **decrease the expression of LHR** and the enzymes **STAR** and **CYP19A1** (important enzymes for the production of progesterone). Another important factor is activin, which is also expressed in the corpus luteum. **Activin** induces functional luteolysis by **reducing LH receptor** levels and the expression of the **STAR** enzyme. If an embryo is present, **HCG prevents the expression of BMP and activin**, protecting the corpus luteum. Luteolysis is divided into two phases: - - The roles of estrogen (ES) and progesterone (PG) are essential during this process. During the follicular phase, estrogen promotes the proliferation of the endometrium. After ovulation, marked by the LH peak, progesterone is secreted, which is crucial for the secretory phase of the menstrual cycle and embryo development. When it comes to embryo transfer, frozen embryos can be transferred during a natural cycle at a specific time. Alternatively, the endometrium can be prepared in advance to optimize conditions for implantation. **STIMULATION FACTS:** 1. 2. 3. 4. In in vitro fertilization (IVF) procedures, we perform follicle aspiration, which means we retrieve the oocyte without needing to rupture the follicular wall. This contrasts with natural ovulation, where the follicular wall does rupture, releasing the oocyte. The same factors that induce this rupture also facilitate cumulus cell expansion, a crucial process for successful fertilization. When conducting IVF rather than intracytoplasmic sperm injection (ICSI), it is essential to create an optimal environment for cumulus expansion. This expansion allows sperm to effectively cross the extracellular matrix, properly capacitate, and interact with signaling molecules within the matrix, thereby enhancing the likelihood of fertilization. In contrast, during ICSI, the direct injection of sperm into the oocyte circumvents the need for this natural expansion, making it less critical. If male infertility is not an issue, IVF is generally preferred. This method allows for a more natural selection process, where the oocyte can "choose" its partner. For example, in Italy, IVF is often performed whenever possible, enabling the oocyte to select the best sperm, as the human eye is not equipped to make that distinction. **[3. MALE REPRODUCTION]** The primordial germ cells migrate from the allantois through the hindgut to the genital ridge, where either the ovary or the testes will develop. The process begins similarly in both sexes, but it diverges due to differences in gene expression. In males, the expression of the SRY/TDY gene triggers differentiation toward testis formation rather than the ovary. This gene initiates a sequence of events that lead to male-specific development, with SRY being the key driver of male differentiation. In females, the migration of cells leads to the formation of ovarian cords, where oogonia proliferate, some of which will regress. The remaining cells form the follicles. These primordial germ cells are guided by other cells to become primordial follicles, and the surrounding cells in females differentiate into granulosa cells. In contrast, in males, these supporting cells differentiate into Sertoli cells, which are crucial for spermatogenesis by nurturing the germ cell line in the testes. Coelomic epithelial cells migrate to form sex cords (granulosa in females, Sertoli in males), while mesonephric cells later contribute to forming theca cells in females and Leydig cells in males. FGF9 plays a crucial role in the differentiation and formation of the seminiferous tubules in males. It is essential for the invasion of mesonephric cells and the development of myoid cells and seminiferous tubules, which are long tubes that coil and condense. Anti-Müllerian hormone (AMH) is expressed in Sertoli cells and is responsible for male sexual differentiation. Together with androgens produced by Leydig cells (which function similarly to theca cells in females), AMH promotes male gonadal development. Leydig cells begin producing testosterone very early, which is crucial for inducing the male phenotype. The small amount of testosterone produced at this stage drives this differentiation. **3.1 MEIOSIS**\  Meiosis in males begins after puberty and proceeds continuously without any pauses during meiosis I. Another unique aspect of male meiosis is that it produces four cells from each spermatogonium, unlike in females where polar bodies are formed. In males, all cells are utilized. The progression is as follows: One key question is why meiosis does not activate before puberty. In males, **aromatase** plays a crucial role by degrading retinoic acid (**RA**), which prevents the activation of meiosis. According to recent models, in the anteroposterior axis, RA stimulates the gene **STRA8**, which is responsible for the activation and induction of meiosis. However, the expression of aromatase degrades RA, blocking meiosis activation. It is only when the steroidogenic pathways change and aromatase is suppressed that meiosis can begin. This is one of the main reasons why meiosis in males does not occur before puberty. - - - Afterward, spermiogenesis occurs in the lumen, where spermatids are transformed into spermatozoa. Even in adulthood, spermatogonia can continue to proliferate and produce four sperm cells, making the process more efficient compared to female meiosis. The seminiferous tubules exhibit morphological irregularities and have a very close association with Sertoli cells, which play a critical role in supporting and nourishing the developing sperm cells. In the testes, spermatogenesis occurs, which is the process of sperm cell development. One major concern in male fertility is **DNA fragmentation**. This issue is particularly relevant when fertility is in question due to age or other factors, as DNA fragmentation can affect the quality of sperm. After being produced, spermatozoa travel through the epididymis, the bladder, and then the seminal vesicle, where various factors are released. The prostate also plays a role in this journey. The epididymis is where sperm is stored, and it is thought that DNA fragmentation mainly occurs here, especially in older men. To reduce DNA fragmentation due to prolonged storage, men are often advised to ejaculate at least two days before sperm collection. This ensures \"fresher\" semen with lower DNA fragmentation levels. Oxidative stress is a natural part of sperm life but is also regulated by important buffering systems. There is a balance: while oxidative stress can be damaging at high levels, it is actually necessary at a moderate level for proper sperm function. In a cross-section of seminiferous tubules, the cells are seen migrating toward the lumen as they differentiate. Less differentiated cells are found at the base. There are three major types of cells within the testes: - - - A key difference between males and females is that, after birth, oogonia in females are all converted into oocytes, whereas males continue to produce sperm throughout life. Leydig cells are not located inside the seminiferous tubules but in the interstitial space between the tubules. The male reproductive system is also influenced by the hypothalamus and pituitary glands, although the release of FSH and LH in males does not follow a cyclic pattern like in females. In males, GnRH stimulates the pituitary to produce FSH (which stimulates Sertoli cells) and LH (which stimulates Leydig cells to produce testosterone). Testosterone is necessary for the paracrine regulation of spermatogenesis. All the factors that affect the female reproductive system also impact males. It is not advisable to give males excess testosterone because it has a negative feedback effect on the hypothalamus. When testosterone levels are too high, they suppress the hypothalamus, reducing FSH and LH levels. In females, increased testosterone can alter GnRH release, raising libido but also reducing follicular development, and excess androgen activity can even lead to breast cancer. For treating fertility problems in males, FSH and LH are often administered. Additionally, managing stress and smoking cessation are crucial. New technologies like microfluidics and AI are also being used to select the best sperm based on their movement and quality. **3.2 SPERMATOGENESIS REGULATION** There is clearly a paracrine regulation of spermatogenesis. Spermatogonia are pluripotent cells, and GDNF (Glial cell line-Derived Neurotrophic Factor) helps maintain spermatogonia in this pluripotent state, ensuring that not all spermatogonia differentiate simultaneously. Activin plays a role in suppressing pluripotency and, when present, can prompt spermatogonia to begin differentiation. BMP4 and KitL (Kit ligand) also promote spermatogonial differentiation, while other BMPs (Bone Morphogenetic Proteins) are important for the initiation of meiosis. While specific factors vary, the key point is the regulation of spermatogenesis by various signals and factors. Leydig cells are positioned around blood vessels to receive oxygen and nutrients necessary for androgen production, including dihydrotestosterone (DHT). Although Sertoli cells can also produce some testosterone, they do so in smaller amounts than Leydig cells. Sertoli cells also produce estradiol. If a man takes external testosterone, it doesn't necessarily mean that there will be sufficient testosterone in the testes. The externally supplied testosterone primarily circulates in the bloodstream and affects muscles, but it doesn't reach the testes in significant amounts because LH is required for local testosterone production in the testes. The high levels of testosterone in the blood create a negative feedback loop, reducing the production of LH and FSH, which in turn reduces local testosterone levels in the testes. This lack of local testosterone can lead to testicular shrinkage, or atrophy. Therefore, it is crucial to produce testosterone locally in the testes for proper spermatogenesis. External testosterone alone cannot adequately supply the paracrine presence of testosterone that is necessary for sperm development. Androgens are essential for maintaining the paracrine environment within the testes. **3.3 SPERMIOGENESIS** Once secondary spermatocytes transition into spermatids, they travel through the seminiferous tubules and gradually develop into spermatozoa. During this process, they acquire several important characteristics: - - - - - - **[PHYSIOLOGY AND ART]** **Advanced maternal age: the greatest challenge in human reproductive medicine** One of the major challenges that patients come to us with is the increased risk of aneuploidy (abnormal number of chromosomes). The image suggests strategies to prevent this, such as using vitrification (freezing eggs) before cancer treatment. Other approaches include using PGT-A (preimplantation genetic testing for aneuploidy) or adapted controlled ovarian stimulation (COS) protocols. However, using oocyte donation as a solution is more of a temporary fix rather than a true solution to the problem. As a woman ages, FSH levels increase. One of the markers indicating the approach of menopause is FSH, alongside AMH (Anti-Müllerian hormone). As the follicle population declines, there is less estradiol produced, which reduces negative feedback, causing FSH levels to rise. This rise in FSH can also serve as a marker for fertility. However, FSH is not the most reliable indicator of fertility. Maternal age remains the most critical factor for fertility compared to FSH levels. AMH, on the other hand, is a more precise indicator of ovarian reserve and the number of growing follicles, making it a better marker for fertility than FSH. The Buratini lab conducted an experiment involving women of different age groups, examining the relationship between FSH and live birth rates, as well as the relationship between AMH and live birth rates. The analysis controlled for all potential variables to determine whether these hormones were independently associated with live birth rates. The results showed that FSH is independently associated with live birth, while AMH was not, which was surprising. The FSH levels measured were basal levels before starting the ovarian stimulation treatment. Interestingly, the number of oocytes retrieved was not associated with FSH levels. While FSH has traditionally been linked to oocyte quantity, these findings suggest that FSH is more related to the quality of oocytes rather than the quantity. Lower FSH levels were associated with higher oocyte quality, indicating that basal FSH is a marker of oocyte quality rather than quantity. The FSH levels being monitored are basal levels measured before starting superovulation treatments. Interestingly, the number of oocytes retrieved was not associated with FSH levels. This means that while FSH has traditionally been linked to the quantity of oocytes, it appears that FSH can predict the chance of a live birth independently of the number of oocytes or oocytes retrieved. These results suggest that basal FSH is not correlated with the number of oocytes produced but rather with their quality. Lower FSH levels are associated with higher-quality oocytes, indicating that FSH influences oocyte quality more than quantity. One puzzling aspect of these findings was the variable association between FSH levels and live births in younger women versus those of advanced maternal age. Through a review of the literature, it was noted that as women age, the variability in FSH levels between cycles increases. While basal FSH levels are typically measured on day 2 of the cycle, this variability makes FSH levels less reliable as a fertility marker in older women. This variability stems from the fact that in some cycles, the dominant follicle from the previous cycle\'s luteal phase can still be regressing during the early follicular phase of the new cycle. This regressing follicle continues to produce inhibin and other negative feedback factors, which can temporarily lower FSH levels at the start of the new cycle. In contrast, women without a clear luteal-phase dominant follicle experience more stable FSH levels. In advanced maternal age patients, the likelihood of finding large, dominant follicles during the luteal phase increases. These follicles can \"invade\" the new cycle's follicular phase, momentarily suppressing FSH levels and making FSH measurements on day 2 less reliable. This explains why FSH is a less dependable fertility marker in older women compared to younger women. The values are not reliable but that doesnt mean that it\`s not important because it regulates the oocyte quality A study investigated individual oocytes and analyzed their correlation with live birth rates, alongside the concentrations of AMH and FSH within the follicles where the oocytes matured. The results showed that the follicular fluid of oocytes that led to live births had higher AMH concentrations and lower FSH concentrations. While superstimulation with FSH does activate follicles, it comes at a cost---it reduces oocyte quality. AMH is a paracrine factor that inhibits FSH. Therefore, a follicle with low FSH and high AMH will have significantly reduced FSH activity. AMH inhibits FSH action, aromatase expression, and estradiol production in granulosa lutein cells. Additionally, it lowers the follicle's sensitivity to FSH in granulosa cells. Excessive FSH activity can lead to granulosa cell apoptosis. It may also reduce communication between granulosa cells and oocytes by decreasing the density of transzonal processes. At the tips of these processes lies a gametic synapse, which sends polyA RNA to the oocyte. This RNA is not only transported but also immediately translated into proteins that are vital for the oocyte, particularly those regulating cytoskeletal dynamics needed for meiosis. In advanced maternal age, higher levels of aneuploidy are observed, raising the question of whether FSH interferes with the cellular connections, contributing to this issue. Since the oocyte cannot metabolize glucose through glycolysis, it depends on pyruvate, NADPH, and PRPP (for nucleotide production) from surrounding cumulus cells. If excessive FSH reduces the communication between the cumulus cells and the oocyte, the result is a decline in oocyte quality. FSH triggers the retraction of transzonal processes through a signaling cascade. In contrast, the oocyte reduces FSH activity in cumulus cells while stimulating AMH, which further suppresses FSH. This suggests that while FSH is necessary, excessive FSH levels during ovarian stimulation can be detrimental. To explore the effects of excessive FSH, researchers divided groups based on different oocyte retrieval numbers. The findings showed that high levels of FSH have a negative impact on live birth rates, highlighting that while FSH is crucial for follicle stimulation, an excessive presence can harm oocyte quality and fertility outcomes. OVARIAN STIMULATION As previously discussed, when FSH stimulation is applied, subordinate follicles continue to grow, and new follicles are recruited, leading to asynchrony among follicles. This means that follicles vary in size, and they are not equivalent in their developmental stages. Preparing the cytoplasm takes time, but one challenge with stimulation is that once the oocyte cytoplasm is ready, it\'s not beneficial for it to wait too long before ovulation. This is one of the complexities of controlled ovarian stimulation (COS). In COS, we use FSH to stimulate follicle growth while simultaneously needing to prevent ovulation. This requires downregulation strategies to suppress the endogenous production of LH and FSH. There are several approaches for achieving this: - - - For GnRH downregulation, we can use GnRH agonists. In the first few days of treatment, agonists initially increase the release of FSH and LH, but after continued administration, the number of GnRH receptors in the gonadal cells decreases, leading to downregulation of FSH and LH production. This helps control the timing of ovulation and prepares the patient for oocyte retrieval in a more controlled manner. Let\'s talk about follicle growth during stimulation. Are we truly following physiological processes in current stimulation protocols? Normally, the final growth of follicles is driven by LH, but many stimulation protocols rely solely on FSH. A new approach suggests introducing LH in the second half of the stimulation process to better mimic natural physiology. For instance, when the leading follicle reaches around 13 mm, some protocols replace half of the FSH dose with LH. However, this is still not fully physiological because a significant amount of FSH remains in use. The idea now is to gradually adjust the ratio of FSH to LH in a way that more closely mimics the body's natural process. By doing this, the protocol may result in fewer oocytes (since some of the smaller, asynchronous follicles would be lost), but the synchrony among the follicles would improve, potentially leading to better oocyte quality. This gradual shift in hormonal support during stimulation could enhance outcomes by better aligning the treatment with natural follicular development, aiming for higher-quality oocytes rather than focusing solely on quantity. Let\'s talk about **poor responsive patients**---those who have difficulty growing multiple follicles during stimulation. There are two types, those who have some mutation and those who have reduced ovarian reserve. A study focused on two groups of patients, all undergoing stimulation. One group (yellow) represented **younger, normal responders**, while the other group (blue) represented **older poor responders**. The study examined the **FSH receptor (FSHR) density** in these patients. Surprisingly, the poor responders seemed to have **more FSH receptors** compared to normal responders. This is counterintuitive because, typically, as ovulation approaches, normal responders lose FSH receptors as part of the **downregulation** process. However, in poor responders, even though they are given higher doses of **FSH** during the stimulation, they maintain higher FSHR levels in larger follicles (around **24 mm**), which is not beneficial. This doesn't result in a higher number of oocytes, and the quality of those oocytes remains poor. So, basically, trying to stimulate poor responders with more FSH does not improve their outcomes, instead it worsens it. **Live birth rates** do not improve. In the other type of **poor responders** with **reduced ovarian reserve** could be caused due to environmental factors. Importantly, the follicles in these patients are quite similar to those of normal responders---they don\'t lack **FSH receptors** and respond to stimulation normally. The key difference is that they simply have fewer follicles. To improve follicle recruitment before starting FSH treatment, we might focus on increasing **follicle transition** and early follicular growth. Several strategies can be employed: 1. 2. 3. For patients with **advanced maternal age** and **AMH levels below 0.75**, this strategy has demonstrated significant improvements. In these cases, the **clinical pregnancy rate** doubled from 10% to 20%, which, while modest, represents a significant improvement. [[Jburatini\@eugin.it]](mailto:[email protected])