التفكير خارج الصندوق في اختبارات الهرمونات

A Broader View of Fertility Assessment: What Standard Tests May Miss

Most IVF clinics begin with a familiar set of “core” labs designed to give a reliable, practical snapshot of ovarian reserve, cycle physiology, and endocrine stability. In women, that usually includes AMH plus early follicular phase gonadotropins and estradiol (FSH, LH, E2), prolactin, and thyroid screening with TSH and free T4. These are sensible starting points, but it is equally important to understand their limits. Ovarian reserve tests are very useful for anticipating how the ovaries may respond to stimulation, yet they are not a direct “fertility score,” and they do not capture many of the physiologic variables that influence egg quality, endometrial receptivity, implantation biology, and how you tolerate or respond to medications (ASRM, 2020). 

That distinction matters most after a disappointing IVF outcome. If standard testing is broadly normal, but the cycle still underperforms (low maturity rate, low fertilization, poor embryo progression, unexpectedly weak response, recurrent biochemical losses, or repeated implantation failure), it can be reasonable to widen the lens. “Thinking outside the box” does not mean ordering every test available. It means choosing a small number of additional markers that are biologically plausible, clinically actionable, and interpreted in context, with clear honesty about what is evidence-based versus what remains evolving. If a test result is not clinically actionable, doing it does not make much sense.

Beyond the basics: micronutrients and metabolic context

Micronutrient status is rarely the single explanation for IVF failure, but it can meaningfully influence reproductive physiology because ovarian function and early embryonic development are energetically demanding and highly sensitive to oxidative stress and inflammatory signaling. Vitamin D is a good example. Vitamin D receptors are present in reproductive tissues, and multiple observational studies and meta-analyses have linked vitamin D sufficiency to better IVF outcomes, though results across studies are not perfectly consistent and causality is difficult to prove (Chu et al., 2018; Hasan et al., 2023). More recently, meta-analytic data on supplementation suggests that, in selected settings, correcting deficiency may improve clinical pregnancy rates, although study design and dosing strategies vary (Meng et al., 2023).

Iron deficiency anemia is increasingly recognized as a clinically relevant but often underappreciated factor influencing fertility in women of reproductive age. Adequate iron availability is essential for ovarian function, folliculogenesis, and early embryonic development, as iron plays a central role in cellular energy metabolism, DNA synthesis, and oxygen transport. Several observational studies have demonstrated that women with iron deficiency, even in the absence of overt anemia, may experience ovulatory dysfunction, luteal phase abnormalities, and reduced conception rates, likely mediated through impaired mitochondrial activity within oocytes and granulosa cells (Cetin et al., 2010; Scholl, 2011). In pregnancy planning populations, low ferritin levels have been associated with prolonged time to pregnancy and poorer outcomes following assisted reproductive technologies, suggesting that iron status may influence both natural and treatment-assisted fertility (Rushton & Barth, 2010; Vujkovic et al., 2010). From a mechanistic standpoint, iron deficiency can exacerbate oxidative stress, disrupt thyroid hormone metabolism, and impair endometrial receptivity, all of which are critical for successful implantation and early placentation (Ganz & Nemeth, 2012).

Elevated homocysteine is also increasingly viewed as a functional marker of impaired one-carbon metabolism with potential downstream effects on fertility in both women and men. From a reproductive perspective, hyperhomocysteinemia is associated with endothelial dysfunction, oxidative stress, and a pro-inflammatory, pro-thrombotic environment, all of which can adversely affect ovarian perfusion, oocyte quality, endometrial receptivity, and early placentation. In women, higher homocysteine levels have been linked to ovulatory dysfunction, poorer embryo quality, reduced implantation rates, and increased risk of early pregnancy loss, particularly in IVF populations, suggesting an effect at both the gamete and implantation level (Vujkovic et al., 2010; Nelen et al., 2000). In men, elevated homocysteine has been associated with increased sperm DNA fragmentation and reduced sperm motility, likely mediated through oxidative damage and impaired methylation-dependent DNA synthesis (Ghorbanian et al., 2016). The most common causes of elevated homocysteine are nutritional deficiencies in folate, vitamin B12, and vitamin B6, which are essential cofactors in homocysteine remethylation and transsulfuration pathways, but renal dysfunction, hypothyroidism, smoking, high alcohol intake, certain medications (including methotrexate, antiepileptics, and metformin), and advancing age also contribute (Refsum et al., 2004; Selhub, 2006). Genetic polymorphisms in enzymes such as MTHFR can modestly raise homocysteine levels, but their clinical relevance depends largely on nutritional status rather than genotype alone.

This is exactly where “outside the box” testing becomes practical: if vitamin D is low or homocysteine is high or if there is iron deficiency, they are easy to correct, safe when done responsibly, and often improves overall health even if it does not single-handedly transform IVF prognosis. The same logic applies to other commonly relevant nutrients that can drift low in real life (especially in restrictive diets, malabsorption, heavy training loads, GLP-1 use or prolonged stress). In these cases, targeted testing and correction can remove avoidable physiologic friction before another cycle. The goal is to identify correctable deficiencies that may interact with ovarian response, mitochondrial function, and inflammation.

Androgens and adrenal contribution: testosterone, DHEA-S, and SHBG

Androgens are often misunderstood in women because they are frequently discussed only in the context of male physiology or PCOS in women. In reality, androgens also have a physiologic role in early follicular development, follicle sensitivity to FSH, and granulosa cell function. In some women, particularly those with diminished ovarian reserve or poor ovarian response, a low-androgen environment can coincide with weaker recruitment and suboptimal stimulation dynamics. This is why some clinicians consider testing total testosterone and DHEA-S along with SHBG (which strongly influences “free” hormone availability), especially after a poor response that is not fully explained by AMH/AFC alone.

What about treatment? DHEA supplementation is widely discussed online, but the scientific picture is mixed: some meta-analyses suggest potential benefit in certain poor-responder or diminished-reserve subgroups, while other higher-quality studies and recent analyses do not show consistent improvement in key outcomes such as mature oocyte yield or live birth (Huang et al., 2025; Conforti et al., 2025). That does not mean DHEA is “useless”; it means it should not be treated as a universal solution. The value of measuring testosterone, DHEA-S, and SHBG is that it can help identify extremes (too low or too high), guide individualized dosing decisions if any androgen modulation is considered, and, importantly, prevent unnecessary supplementation in women who already have adequate levels.

Hormone metabolites: when “how you process hormones” may matter

Standard serum labs measure the amount of a hormone circulating in blood at one moment. That is often sufficient, but it is not always the full story. Hormone metabolites measured in urine can offer a different layer of information: patterns of hormone production over time, diurnal variation (for some hormones), and downstream metabolism. Urinary methods have been studied as practical surrogates for reproductive hormone monitoring and can reflect overall hormonal output across the day when appropriately collected (Newman et al., 2019; Newman et al., 2021). 

Where this can become clinically meaningful is not in “diagnosing fertility” from metabolites, but in solving specific puzzles. For example, if luteal phase support seems consistently inadequate despite standard progesterone strategies, if estrogen exposure appears unusually high or low relative to stimulation response, or if symptoms and cycle behavior do not fit the serum snapshot, metabolite patterns may provide clues that help tailor timing or dosing. For instance, if a patient preferentially metabolizes estrogen toward 4-hydroxyestrone, this can be clinically relevant in fertility treatment because 4-OH estrone is a reactive estrogen metabolite with limited physiologic estrogenic support and a higher potential for oxidative stress at the tissue level. In this setting, total estrogen levels may appear sufficient, yet the balance of estrogen metabolites may be less favorable for optimal endometrial maturation and cellular signaling

5-point cortisol testing: the HPA axis and reproductive resilience

Stress is real, infertility itself is stressful, and the HPA axis influences reproductive signaling but it is also true that many people conceive and succeed with IVF during stressful life periods. When we look specifically at cortisol, the evidence linking cortisol levels to IVF outcomes is inconsistent. Systematic reviews show mixed associations, often limited by study quality, timing of sampling, and confounders (Massey et al., 2014; Karunyam et al., 2023). Some recent reviews conclude that acute or perceived stress does not reliably predict IVF outcomes at key procedural stages, even when cortisol is measured (Zanettoullis et al., 2024). 

So why would a clinic ever consider multi-point cortisol testing? Because the question is not always “does stress cause IVF failure?” Sometimes the question is more practical: is there a pattern of HPA axis dysregulation (flattened diurnal rhythm, exaggerated evening cortisol, or unusually low morning cortisol) that correlates with sleep disruption, fatigue, anxiety symptoms, blood sugar instability, or inflammatory patterns that may reduce physiologic resilience during stimulation and early pregnancy? A 4- or 5-point cortisol profile can sometimes help structure an individualized plan focused on sleep timing, light exposure, exercise dosing, nutrition, and stress physiology. It is not a fertility “decoder,” but in selected patients it can support a more coherent, physiology-driven optimization plan, particularly when symptoms and lifestyle context strongly suggest HPA strain.

For thyroid-treated patients: fT3 can help

Thyroid function matters in reproduction, and thyroid dysfunction is associated with menstrual irregularities and adverse pregnancy outcomes (Poppe et al., 2007; Brown et al., 2023). That is why TSH and free T4 are standard in IVF workups. In patients already on thyroid hormone, however, additional nuance sometimes matters. Measuring free T3 can occasionally be useful when symptoms, basal temperature patterns, or metabolic features suggest that T4-to-T3 conversion may be suboptimal, especially if TSH looks “fine” but the patient feels unwell. That said, the more controversial marker is reverse T3 (rT3). rT3 can show us if fT4 is converted into the inactive form of T3 rather than the active form. This can show us whether additional T3 supplementation should be considered or not. While this is not a standard test, it can be incorporated into the testing regimen of patients who are already on thyroid replacement therapy and still feel unwell.

The real point of “non-standard” tests is actionability, not curiosity!

The purpose of expanding testing is not to generate more numbers; it is to uncover patterns that can change decisions. In a carefully selected patient, these tests can help us personalize stimulation strategy, address correctable deficiencies, avoid unnecessary supplements, improve medication tolerability, and optimize the physiologic environment for egg development and early pregnancy. At the same time, it is important to be honest: no expanded panel can guarantee success, and many IVF failures happen because of embryo genetics that no blood test can fully predict in advance. Even the best testing strategy is simply a way to reduce avoidable blind spots and tailor the plan more intelligently than a one-size-fits-all approach.

If you have already been through an unsuccessful IVF cycle and your standard assessment doesn’t explain what happened, a “thinking outside the box” approach can be a rational next step provided it is selective, evidence-based, and focused on interventions that are realistic and safe.

د. احمد اوزيجيت, MD, ABAARM
أخصائي مكافحة الشيخوخة والطب التجديدي
عالم الأجنة السريري